KC Huang

All Publications

• Pictures of Tongues Sticking Out. Trends in endocrinology and metabolism: TEM Shi, H., Huang, K. C. 2020

  Abstract

  Despite their small sizes, bacterial cells within a host-associated microbial community often form highly structured complexes determined by environmental factors and interspecies interactions. Wilbert et al. combined species-specific fluorescent labels and high-resolution microscopy to visualize human tongue dorsum microbiomes and to highlight their structure and dynamics.

  View details for DOI 10.1016/j.tem.2020.05.003

  View details for PubMedID 32475653

• Colons or semi-colons: punctuating the regional variation of intestinal microbial-immune interactions. Nature reviews. Gastroenterology & hepatology Culver, R. N., Spencer, S. P., Huang, K. C. 2020

  View details for DOI 10.1038/s41575-020-0302-z

  View details for PubMedID 32322050

• Chiral twisting in a bacterial cytoskeletal polymer affects filament size and orientation. Nature communications Shi, H., Quint, D. A., Grason, G. M., Gopinathan, A., Huang, K. C. 2020; 11 (1): 1408

  Abstract

  In many rod-shaped bacteria, the actin homolog MreB directs cell-wall insertion and maintains cell shape, but it remains unclear how structural changes to MreB affect its organization in vivo. Here, we perform molecular dynamics simulations for Caulobacter crescentus MreB to extract mechanical parameters for inputs into a coarse-grained biophysical polymer model that successfully predicts MreB filament properties in vivo. Our analyses indicate that MreB double protofilaments can exhibit left-handed twisting that is dependent on the bound nucleotide and membrane binding; the degree of twisting correlates with the length and orientation of MreB filaments observed in vitro and in vivo. Our molecular dynamics simulations also suggest that membrane binding of MreB double protofilaments induces a stable membrane curvature of similar magnitude to that observed in vivo. Thus, our multiscale modeling correlates cytoskeletal filament size with conformational changes inferred from molecular dynamics simulations, providing a paradigm for connecting protein filament structure and mechanics to cellular organization and function.

  View details for DOI 10.1038/s41467-020-14752-9

  View details for PubMedID 32179732

• Bacterial interspecies interactions modulate pH-mediated antibiotic tolerance. eLife Aranda-Diaz, A., Obadia, B., Dodge, R., Thomsen, T., Hallberg, Z. F., Guvener, Z. T., Ludington, W. B., Huang, K. C. 2020; 9

  Abstract

  Predicting antibiotic efficacy within microbial communities remains highly challenging. Interspecies interactions can impact antibiotic activity through many mechanisms, including alterations to bacterial physiology. Here, we studied synthetic communities constructed from the core members of the fruit fly gut microbiota. Co-culturing of Lactobacillus plantarum with Acetobacter species altered its tolerance to the transcriptional inhibitor rifampin. By measuring key metabolites and environmental pH, we determined that Acetobacter species counter the acidification driven by L. plantarum production of lactate. Shifts in pH were sufficient to modulate L. plantarum tolerance to rifampin and the translational inhibitor erythromycin. A reduction in lag time exiting stationary phase was linked to L. plantarum tolerance to rifampicin, opposite to a previously identified mode of tolerance to ampicillin in E. coli. This mechanistic understanding of the coupling among interspecies interactions, environmental pH, and antibiotic tolerance enables future predictions of growth and the effects of antibiotics in more complex communities.

  View details for DOI 10.7554/eLife.51493

  View details for PubMedID 31995029

• Klebsiella michiganensis transmission enhances resistance to Enterobacteriaceae gut invasion by nutrition competition. Nature microbiology Oliveira, R. A., Ng, K. M., Correia, M. B., Cabral, V., Shi, H., Sonnenburg, J. L., Huang, K. C., Xavier, K. B. 2020

  Abstract

  Intestinal microbiotas contain beneficial microorganisms that protect against pathogen colonization; treatment with antibiotics disrupts the microbiota and compromises colonization resistance. Here, we determine the impact of exchanging microorganisms between hosts on resilience to the colonization of invaders after antibiotic-induced dysbiosis. We assess the functional consequences of dysbiosis using a mouse model of colonization resistance against Escherichia coli. Antibiotics caused stochastic loss of members of the microbiota, but the microbiotas of co-housed mice remained more similar to each other compared with the microbiotas among singly housed animals. Strikingly, co-housed mice maintained colonization resistance after treatment with antibiotics, whereas most singly housed mice were susceptible to E. coli. The ability to retain or share the commensal Klebsiella michiganensis, a member of the Enterobacteriaceae family, was sufficient for colonization resistance after treatment with antibiotics. K. michiganensis generally outcompeted E. coli in vitro, but in vivo administration of galactitol-a nutrient that supports the growth of only E. coli-to bi-colonized gnotobiotic mice abolished the colonization-resistance capacity of K. michiganensis against E. coli, supporting the idea that nutrient competition is the primary interaction mechanism. K. michiganensis also hampered colonization of the pathogen Salmonella, prolonging host survival. Our results address functional consequences of the stochastic effects of microbiota perturbations, whereby microbial transmission through host interactions can facilitate reacquisition of beneficial commensals, minimizing the negative impact of antibiotics.

  View details for DOI 10.1038/s41564-019-0658-4

  View details for PubMedID 31959968

• Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila. PLoS biology Koyama, L. A., Aranda-Díaz, A. n., Su, Y. H., Balachandra, S. n., Martin, J. L., Ludington, W. B., Huang, K. C., O'Brien, L. E. 2020; 18 (1): e3000567

  Abstract

  Cell- and tissue-level processes often occur across days or weeks, but few imaging methods can capture such long timescales. Here, we describe Bellymount, a simple, noninvasive method for longitudinal imaging of the Drosophila abdomen at subcellular resolution. Bellymounted animals remain live and intact, so the same individual can be imaged serially to yield vivid time series of multiday processes. This feature opens the door to longitudinal studies of Drosophila internal organs in their native context. Exploiting Bellymount's capabilities, we track intestinal stem cell lineages and gut microbial colonization in single animals, revealing spatiotemporal dynamics undetectable by previously available methods.

  View details for DOI 10.1371/journal.pbio.3000567

  View details for PubMedID 31986129

• AimB Is a Small Protein Regulator of Cell Size and MreB Assembly. Biophysical journal Werner, J. N., Shi, H. n., Hsin, J. n., Huang, K. C., Gitai, Z. n., Klein, E. A. 2020

  Abstract

  The MreB actin-like cytoskeleton assembles into dynamic polymers that coordinate cell shape in many bacteria. In contrast to most other cytoskeleton systems, few MreB-interacting proteins have been well characterized. Here, we identify a small protein from Caulobacter crescentus, an assembly inhibitor of MreB (AimB). AimB overexpression mimics inhibition of MreB polymerization, leading to increased cell width and MreB delocalization. Furthermore, aimB appears to be essential, and its depletion results in decreased cell width and increased resistance to A22, a small-molecule inhibitor of MreB assembly. Molecular dynamics simulations suggest that AimB binds MreB at its monomer-monomer protofilament interaction cleft and that this interaction is favored for C. crescentus MreB over Escherichia coli MreB because of a closer match in the degree of opening with AimB size, suggesting coevolution of AimB with MreB conformational dynamics in C. crescentus. We support this model through functional analysis of point mutants in both AimB and MreB, photo-cross-linking studies with site-specific unnatural amino acids, and species-specific activity of AimB. Together, our findings are consistent with AimB promoting MreB dynamics by inhibiting monomer-monomer assembly interactions, representing a new mechanism for regulating actin-like polymers and the first identification of a non-toxin MreB assembly inhibitor. Because AimB has only 104 amino acids and small proteins are often poorly characterized, our work suggests the possibility of more bacterial cytoskeletal regulators to be found in this class. Thus, like FtsZ and eukaryotic actin, MreB may have a rich repertoire of regulators to tune its precise assembly and dynamics.

  View details for DOI 10.1016/j.bpj.2020.04.029

  View details for PubMedID 32416080

• Biosurfactant-Mediated Membrane Depolarization Maintains Viability during Oxygen Depletion in Bacillus subtilis. Current biology : CB Arjes, H. A., Vo, L. n., Dunn, C. M., Willis, L. n., DeRosa, C. A., Fraser, C. L., Kearns, D. B., Huang, K. C. 2020

  Abstract

  The presence or absence of oxygen in the environment is a strong effector of cellular metabolism and physiology. Like many eukaryotes and some bacteria, Bacillus subtilis primarily utilizes oxygen during respiration to generate ATP. Despite the importance of oxygen for B. subtilis survival, we know little about how populations adapt to shifts in oxygen availability. Here, we find that when oxygen was depleted from stationary phase B. subtilis cultures, ∼90% of cells died while the remaining cells maintained colony-forming ability. We discover that production of the antimicrobial surfactin confers two oxygen-related fitness benefits: it increases aerobic growth yield by increasing oxygen diffusion, and it maintains viability during oxygen depletion by depolarizing the membrane. Strains unable to produce surfactin exhibited an ∼50-fold reduction in viability after oxygen depletion. Surfactin treatment of these cells led to membrane depolarization and reduced ATP production. Chemical and genetic perturbations that alter oxygen consumption or redox state support a model in which surfactin-mediated membrane depolarization maintains viability through slower oxygen consumption and/or a shift to a more reduced metabolic profile. These findings highlight the importance of membrane potential in regulating cell physiology and growth, and demonstrate that antimicrobials that depolarize cell membranes can benefit cells when the terminal electron acceptor in respiration is limiting. This foundational knowledge has deep implications for environmental microbiology, clinical anti-bacterial therapy, and industrial biotechnology.

  View details for DOI 10.1016/j.cub.2020.01.073

  View details for PubMedID 32059765

• Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila PLOS BIOLOGY Koyama, L., Aranda-Diaz, A., Su, Y., Balachandra, S., Martin, J., Ludington, W. B., Huang, K., O'Brien, L. 2020; 18 (1)

  View details for DOI 10.1371/journal.pbio.3000567.r004

  View details for Web of Science ID 000510743800005

• FtsZ-Independent Mechanism of Division Inhibition by the Small Molecule PC190723 in Escherichia coli ADVANCED BIOSYSTEMS Khare, S., Hsin, J., Sorto, N. A., Nepomuceno, G. M., Shaw, J. T., Shi, H., Huang, K. 2019; 3 (11)

  View details for DOI 10.1002/adbi.201900021

  View details for Web of Science ID 000498192300009

• Decoupling of Rates of Protein Synthesis from Cell Expansion Leads to Supergrowth. Cell systems Knapp, B. D., Odermatt, P., Rojas, E. R., Cheng, W., He, X., Huang, K. C., Chang, F. 2019

  Abstract

  Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis or the consequences when this homeostasis is perturbed. Here, we identify conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to general accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of "supergrowth" wherein rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a mechanism for global proteome homeostasis based on modulation of volume growth and dilution.

  View details for DOI 10.1016/j.cels.2019.10.001

  View details for PubMedID 31706948

• Mechanically resolved imaging of bacteria using expansion microscopy. PLoS biology Lim, Y., Shiver, A. L., Khariton, M., Lane, K. M., Ng, K. M., Bray, S. R., Qin, J., Huang, K. C., Wang, B. 2019; 17 (10): e3000268

  Abstract

  Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (muExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of muExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

  View details for DOI 10.1371/journal.pbio.3000268

  View details for PubMedID 31622337

• Chromosome Organization: Making Room in a Crowd. Current biology : CB Shi, H., Huang, K. C. 2019; 29 (13): R630–R632

  Abstract

  Despite their small size and lack of membrane-based DNA encapsulation, prokaryotic cells still organize and scale their nucleoid in specific subcellular regions. Two studies show that the DNA-free regions in prokaryotes are full of large biomolecules, which exclude DNA via entropic forces.

  View details for DOI 10.1016/j.cub.2019.06.002

  View details for PubMedID 31287980

• tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance CELL SYSTEMS Masuda, I., Matsubara, R., Christian, T., Rojas, E. R., Yadavalli, S. S., Zhang, L., Goulian, M., Foster, L., Huang, K., Hou, Y. 2019; 8 (4): 302-+

  View details for DOI 10.1016/j.cels.2019.03.008

  View details for Web of Science ID 000465541100004

• tRNA Methylation Is a Global Determinant of Bacterial Multi-drug Resistance. Cell systems Masuda, I., Matsubara, R., Christian, T., Rojas, E. R., Yadavalli, S. S., Zhang, L., Goulian, M., Foster, L., Huang, K. C., Hou, Y. 2019

  Abstract

  Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show thatm1G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m1G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m1G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.

  View details for PubMedID 30981730

• Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus MOLECULAR MICROBIOLOGY Stankeviciute, G., Miguel, A. V., Radkov, A., Chou, S., Huang, K., Klein, E. A. 2019; 111 (4): 995–1008

  View details for DOI 10.1111/mmi.14199

  View details for Web of Science ID 000464655800010

• Conservation of conformational dynamics across prokaryotic actins. PLoS computational biology Ng, N., Shi, H., Colavin, A., Huang, K. C. 2019; 15 (4): e1006683

  Abstract

  The actin family of cytoskeletal proteins is essential to the physiology of virtually all archaea, bacteria, and eukaryotes. While X-ray crystallography and electron microscopy have revealed structural homologies among actin-family proteins, these techniques cannot probe molecular-scale conformational dynamics. Here, we use all-atom molecular dynamic simulations to reveal conserved dynamical behaviors in four prokaryotic actin homologs: MreB, FtsA, ParM, and crenactin. We demonstrate that the majority of the conformational dynamics of prokaryotic actins can be explained by treating the four subdomains as rigid bodies. MreB, ParM, and FtsA monomers exhibited nucleotide-dependent dihedral and opening angles, while crenactin monomer dynamics were nucleotide-independent. We further show that the opening angle of ParM is sensitive to a specific interaction between subdomains. Steered molecular dynamics simulations of MreB, FtsA, and crenactin dimers revealed that changes in subunit dihedral angle lead to intersubunit bending or twist, suggesting a conserved mechanism for regulating filament structure. Taken together, our results provide molecular-scale insights into the nucleotide and polymerization dependencies of the structure of prokaryotic actins, suggesting mechanisms for how these structural features are linked to their diverse functions.

  View details for PubMedID 30951524

• Conservation of conformational dynamics across prokaryotic actins PLOS COMPUTATIONAL BIOLOGY Ng, N., Shi, H., Colavin, A., Huang, K. 2019; 15 (4)

  View details for DOI 10.1371/journal.pcbi.1006683

  View details for Web of Science ID 000467530600016

• Conformational Dynamics of a Bacterial Actin Filament Predict In Vivo Filament Length Huang, K. C. CELL PRESS. 2019: 5A

  View details for DOI 10.1016/j.bpj.2018.11.053

  View details for Web of Science ID 000460779800025

• Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus. Molecular microbiology Stankeviciute, G., Miguel, A. V., Radkov, A., Chou, S., Huang, K. C., Klein, E. A. 2019

  Abstract

  The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod-shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme-resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell-body peptidoglycan contained primarily DD-crosslinks between meso-diaminopimelic acid and D-alanine residues, whereas stalk peptidoglycan had more LD-transpeptidation (meso-diaminopimelic acid-meso-diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD-transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD-crosslinking. Despite no obvious selection pressure for LD-crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP-DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30614079

• Cell geometry and leaflet bilayer asymmetry regulate domain formation in plasma membranes PHYSICAL REVIEW E Ali, M., Huang, K., Wingreen, N. S., Mukhopadhyay, R. 2019; 99 (1)

  View details for DOI 10.1103/PhysRevE.99.012401

  View details for Web of Science ID 000454769800009

• Cell geometry and leaflet bilayer asymmetry regulate domain formation in plasma membranes. Physical review. E Ali, M. Z., Huang, K. C., Wingreen, N. S., Mukhopadhyay, R. 2019; 99 (1-1): 012401

  Abstract

  We model how pattern formation in a multicomponent lipid bilayer pinned to an elastic substrate is governed by the interplay between lipid phase separation and the tendency of domains of high intrinsic curvature lipids to deform the membrane away from a stiff substrate such as the cell wall. The emergent patterns, which include compact and striped lipid microdomains, are anticorrelated across the two leaflets and depend on leaflet asymmetry, the ability of lipids to flip between leaflets, and the global geometry. We characterize analytically the dependence of stripe width on lipid parameters, and consider the implications of interleaflet patterning for curvature-dependent lipid localization.

  View details for DOI 10.1103/PhysRevE.99.012401

  View details for PubMedID 30780246

• Recovery of the Gut Microbiota after Antibiotics Depends on Host Diet, Community Context, and Environmental Reservoirs. Cell host & microbe Ng, K. M., Aranda-Díaz, A. n., Tropini, C. n., Frankel, M. R., Van Treuren, W. n., O'Laughlin, C. T., Merrill, B. D., Yu, F. B., Pruss, K. M., Oliveira, R. A., Higginbottom, S. K., Neff, N. F., Fischbach, M. A., Xavier, K. B., Sonnenburg, J. L., Huang, K. C. 2019; 26 (5): 650–65.e4

  Abstract

  Antibiotics alter microbiota composition and increase infection susceptibility. However, the generalizable effects of antibiotics on and the contribution of environmental variables to gut commensals remain unclear. To address this, we tracked microbiota dynamics with high temporal and taxonomic resolution during antibiotic treatment in a controlled murine system by isolating variables such as diet, treatment history, and housing co-inhabitants. Human microbiotas were remarkably resilient and recovered during antibiotic treatment, with transient dominance of resistant Bacteroides and taxa-asymmetric diversity reduction. In certain cases, in vitro sensitivities were not predictive of in vivo responses, underscoring the significance of host and community context. A fiber-deficient diet exacerbated microbiota collapse and delayed recovery. Species replacement through cross housing after ciprofloxacin treatment established resilience to a second treatment. Single housing drastically disrupted recovery, highlighting the importance of environmental reservoirs. Our findings highlight deterministic microbiota adaptations to perturbations and the translational potential for modulating diet, sanitation, and microbiota composition during antibiotics.

  View details for DOI 10.1016/j.chom.2019.10.011

  View details for PubMedID 31726029

• When a physicist wanders into biology: an interview with KC Huang. BMC biology Huang, K. C. 2018; 16 (1): 130

  Abstract

  Kerwyn Casey ("KC") Huang is an Associate Professor at Stanford University, studying the physical nature of biological systems and the underpinnings of fundamental processes such as cell shape determination, cell division, and intracellular and microbial community organization. In this interview, KC discusses how the ability to pursue insights at scales from molecules to cellular communities can shed new light on longstanding questions, the necessity for new tools in exploring the microbiome, how to create an empowering lab environment, and why integrating chemistry with physics and biology can bring us closer to asking the right questions.

  View details for PubMedID 30382844

• Who's Your DadA? d-Alanine Levels Regulate Bacterial Stiffness. mBio Odermatt, P. D., Arjes, H. A., Chang, F., Huang, K. C. 2018; 9 (5)

  Abstract

  A central question in mechanobiology is how cellular-scale structures are established and regulated. In bacteria, the cell envelope is essential for mechanical integrity, protecting against environmental stresses and bearing the load from high turgor pressures. Trivedi et al. (mBio 9:e01340-18, 2018, https://doi.org/10.1128/mBio.01340-18) screened a Pseudomonas aeruginosa transposon library and identified genes that influence cell stiffness by measuring cell growth while cells were embedded in an agarose gel. Their findings provide a broad knowledge base for how biochemical pathways regulate cellular mechanical properties in this pathogen. Dozens of genes across diverse functional categories were implicated, suggesting that cellular mechanics is a systems-level emergent property. Furthermore, changes in d-alanine levels in a dadA (d-alanine dehydrogenase) mutant resulted in decreases in the expression of cell wall enzymes, cross-linking density, and cell stiffness. These insights into the biochemical and mechanical roles of dadA highlight the importance of systems-level investigations into the physical properties of cells.

  View details for PubMedID 30352940

• A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection. Cell host & microbe Jacobson, A., Lam, L., Rajendram, M., Tamburini, F., Honeycutt, J., Pham, T., Van Treuren, W., Pruss, K., Stabler, S. R., Lugo, K., Bouley, D. M., Vilches-Moure, J. G., Smith, M., Sonnenburg, J. L., Bhatt, A. S., Huang, K. C., Monack, D. 2018

  Abstract

  The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth invitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S.Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.

  View details for PubMedID 30057174

• The outer membrane is an essential load-bearing element in Gram-negative bacteria. Nature Rojas, E. R., Billings, G., Odermatt, P. D., Auer, G. K., Zhu, L., Miguel, A., Chang, F., Weibel, D. B., Theriot, J. A., Huang, K. C. 2018

  Abstract

  Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

  View details for PubMedID 30022160

• Translating the Physical Code of Life. Cell Knapp, B. D., Huang, K. C. 2018; 174 (2): 253–55

  Abstract

  The cytoplasm is a highly crowded and complex environment, and the regulation of its physical properties has only recently begun to be revealed. In this issue of Cell, Delarue etal. demonstrate that the control of ribosome concentration through mTORC1 sets limits on the diffusion of large particles and controls phase separation in eukaryotic cells.

  View details for PubMedID 30007414

• Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota. Cell Tropini, C., Moss, E. L., Merrill, B. D., Ng, K. M., Higginbottom, S. K., Casavant, E. P., Gonzalez, C. G., Fremin, B., Bouley, D. M., Elias, J. E., Bhatt, A. S., Huang, K. C., Sonnenburg, J. L. 2018; 173 (7): 1742

  Abstract

  Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains invitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.

  View details for PubMedID 29906449

• Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota CELL Tropini, C., Moss, E., Merrill, B., Ng, K., Higginbottom, S., Casavant, E., Gonzalez, C., Fremin, B., Bouley, D., Elias, J., Bhatt, A., Huang, K., Sonnenburg, J. 2018; 173 (7): 1742-+

  View details for DOI 10.1016/j.cell.2018.05.008

  View details for Web of Science ID 000437004000021

• Lateral interactions between protofilaments of the bacterial tubulin homolog FtsZ are essential for cell division ELIFE Guan, F., Yu, J., Yu, J., Liu, Y., Li, Y., Feng, X., Huang, K., Chang, Z., Ye, S. 2018; 7

  Abstract

  The prokaryotic tubulin homolog FtsZ polymerizes into protofilaments, which further assemble into higher-order structures at future division sites to form the Z-ring, a dynamic structure essential for bacterial cell division. The precise nature of interactions between FtsZ protofilaments that organize the Z-ring and their physiological significance remain enigmatic. In this study, we solved two crystallographic structures of a pair of FtsZ protofilaments, and demonstrated that they assemble in an antiparallel manner through the formation of two different inter-protofilament lateral interfaces. Our in vivo photocrosslinking studies confirmed that such lateral interactions occur in living cells, and disruption of the lateral interactions rendered cells unable to divide. The inherently weak lateral interactions enable FtsZ protofilaments to self-organize into a dynamic Z-ring. These results have fundamental implications for our understanding of bacterial cell division and for developing antibiotics that target this key process.

  View details for PubMedID 29889022

• Cutting the Gordian Knot of the Microbiota MOLECULAR CELL Vasquez, K. S., Shiver, A. L., Huang, K. 2018; 70 (5): 765–67

  Abstract

  The gut microbiota plays a central role in human health. Studies by Tramontano et al. (2018) and Maier et al. (2018) improve our understanding of the metabolism and pharmaceutical impact of human gut bacteria through high-throughput screening of growth in the presence of different nutrients and drugs, respectively.

  View details for PubMedID 29883604

• Regulation of microbial growth by turgor pressure CURRENT OPINION IN MICROBIOLOGY Rojas, E. R., Huang, K. 2018; 42: 62–70

  Abstract

  Rapid changes in environmental osmolarity are a natural aspect of microbial lifestyles. The change in turgor pressure resulting from an osmotic shock alters the mechanical forces within the cell envelope, and can impact cell growth across a range of timescales, through a variety of mechanical mechanisms. Here, we first summarize measurements of turgor pressure in various organisms. We then review how the combination of microfluidic flow cells and quantitative image analysis has driven discovery of the diverse ways in which turgor pressure mechanically regulates bacterial growth, independent of the effect of cytoplasmic crowding. In Gram-positive, rod-shaped bacteria, reductions in turgor pressure cause decreased growth rate. Moreover, a hypoosmotic shock, which increases turgor pressure and membrane tension, leads to transient inhibition of cell-wall growth via electrical depolarization. By contrast, Gram-negative Escherichia coli is remarkably insensitive to changes in turgor. We discuss the extent to which turgor pressure impacts processes such as cell division that alter cell shape, in particular that turgor facilitates millisecond-scale daughter-cell separation in many Actinobacteria and eukaryotic fission yeast. This diverse set of responses showcases the potential for using osmotic shocks to interrogate how mechanical perturbations affect cellular processes.

  View details for PubMedID 29125939

• RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape NATURE COMMUNICATIONS Colavin, A., Shi, H., Huang, K. 2018; 9: 1280

  Abstract

  In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape, although the molecular mechanism by which cell width is regulated remains unknown. Here we demonstrate that the membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization of E. coli cell-wall growth. The relative expression levels of MreB and RodZ change in a manner commensurate with variations in growth rate and cell width, and RodZ systematically alters the curvature-based localization of MreB and cell width in a concentration-dependent manner. We identify MreB mutants that alter the bending properties of MreB filaments in molecular dynamics simulations similar to RodZ binding, and show that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Thus, E. coli can control its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.

  View details for PubMedID 29599448

• How to Build a Bacterial Cell: MreB as the Foreman of E. coli Construction CELL Shi, H., Bratton, B. P., Gitai, Z., Huang, K. 2018; 172 (6): 1294–1305

  Abstract

  Cell shape matters across the kingdoms of life, and cells have the remarkable capacity to define and maintain specific shapes and sizes. But how are the shapes of micron-sized cells determined from the coordinated activities of nanometer-sized proteins? Here, we review general principles that have surfaced through the study of rod-shaped bacterial growth. Imaging approaches have revealed that polymers of the actin homolog MreB play a central role. MreB both senses and changes cell shape, thereby generating a self-organizing feedback system for shape maintenance. At the molecular level, structural and computational studies indicate that MreB filaments exhibit tunable mechanical properties that explain their preference for certain geometries and orientations along the cylindrical cell body. We illustrate the regulatory landscape of rod-shape formation and the connectivity between cell shape, cell growth, and other aspects of cell physiology. These discoveries provide a framework for future investigations into the architecture and construction of microbes.

  View details for PubMedID 29522748

  View details for PubMedCentralID PMC5846203

• Membrane Tension Inhibits Wall Synthesis via Electrical Depolarization to Balance Bacterial Cell Envelope Expansion Huang, K., Rojas, E., Theriot, J. CELL PRESS. 2018: 28A

  View details for Web of Science ID 000429315800147

• Locked Expansion Microscopy to in Situ Analyze Microbial Communities Lim, Y., Khariton, M., Bray, S., Ng, K., Shiver, A., Huang, K. C., Wang, B. CELL PRESS. 2018: 532A

  View details for Web of Science ID 000430563200415

• Translational Reprogramming in Salmonella typhimurium Modifies Environmental pH to Sustain Higher Growth Rates before Entry into Stationary Phase Rajendram, M., Zhu, L., Huang, K. C. CELL PRESS. 2018: 664A

  View details for Web of Science ID 000430563300313

• Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 2018; 562 (7727): 367–72

  Abstract

  Here we present a compendium of single-cell transcriptomic data from the model organism Mus musculus that comprises more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, reveal gene expression in poorly characterized cell populations and enable the direct and controlled comparison of gene expression in cell types that are shared between tissues, such as T lymphocytes and endothelial cells from different anatomical locations. Two distinct technical approaches were used for most organs: one approach, microfluidic droplet-based 3'-end counting, enabled the survey of thousands of cells at relatively low coverage, whereas the other, full-length transcript analysis based on fluorescence-activated cell sorting, enabled the characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

  View details for DOI 10.1038/s41586-018-0590-4

  View details for PubMedID 30283141

• Marine Mammal Microbiota Yields Novel Antibiotic with Potent Activity Against Clostridium difficile ACS INFECTIOUS DISEASES Ochoa, J. L., Sanchez, L. M., Koo, B., Doherty, J. S., Rajendram, M., Huang, K., Gross, C. A., Linington, R. G. 2018; 4 (1): 59–67

  Abstract

  The recent explosion of research on the microbiota has highlighted the important interplay between commensal microorganisms and the health of their cognate hosts. Metabolites isolated from commensal bacteria have been demonstrated to possess a range of antimicrobial activities, and it is widely believed that some of these metabolites modulate host behavior, affecting predisposition to disease and pathogen invasion. Our access to the local marine mammal stranding network and previous successes in mining the fish microbiota poised us to test the hypothesis that the marine mammal microbiota is a novel source of commensal bacteria-produced bioactive metabolites. Examination of intestinal contents from five marine mammals led to the identification of a Micromonospora strain with potent and selective activity against a panel of Gram-positive pathogens and no discernible human cytotoxicity. Compound isolation afforded a new complex glycosylated polyketide, phocoenamicin, with potent activity against the intestinal pathogen Clostridium difficile, an organism challenging to treat in hospital settings. Use of our activity-profiling platform, BioMAP, clustered this metabolite with other known ionophore antibiotics. Fluorescence imaging and flow cytometry confirmed that phocoenamicin is capable of shifting membrane potential without damaging membrane integrity. Thus, exploration of gut microbiota in hosts from diverse environments can serve as a powerful strategy for the discovery of novel antibiotics against human pathogens.

  View details for PubMedID 29043783

• Homeostatic Cell Growth Is Accomplished Mechanically through Membrane Tension Inhibition of Cell-Wall Synthesis CELL SYSTEMS Rojas, E. R., Huang, K., Theriot, J. A. 2017; 5 (6): 578-+

  Abstract

  Feedback mechanisms are required to coordinate balanced synthesis of subcellular components during cell growth. However, these coordination mechanisms are not apparent at steady state. Here, we elucidate the interdependence of cell growth, membrane tension, and cell-wall synthesis by observing their rapid re-coordination after osmotic shocks in Gram-positive bacteria. Single-cell experiments and mathematical modeling demonstrate that mechanical forces dually regulate cell growth: while turgor pressure produces mechanical stress within the cell wall that promotes its expansion through wall synthesis, membrane tension induces growth arrest by inhibiting wall synthesis. Tension inhibition occurs concurrently with membrane depolarization, and depolarization arrested growth independently of shock, indicating that electrical signals implement the negative feedback characteristic of homeostasis. Thus, competing influences of membrane tension and cell-wall mechanical stress on growth allow cells to rapidly correct for mismatches between membrane and wall synthesis rates, ensuring balanced growth.

  View details for PubMedID 29203279

  View details for PubMedCentralID PMC5985661

• Plasmon-actuated nano-assembled microshells SCIENTIFIC REPORTS Quint, M. T., Sarang, S., Quint, D. A., Keshavarz, A., Stokes, B. J., Subramaniam, A., Huang, K., Gopinathan, A., Hirst, L. S., Ghosh, S. 2017; 7: 17788

  Abstract

  We present three-dimensional microshells formed by self-assembly of densely-packed 5 nm gold nanoparticles (AuNPs). Surface functionalization of the AuNPs with custom-designed mesogenic molecules drives the formation of a stable and rigid shell wall, and these unique structures allow encapsulation of cargo that can be contained, virtually leakage-free, over several months. Further, by leveraging the plasmonic response of AuNPs, we can rupture the microshells using optical excitation with ultralow power (<2 mW), controllably and rapidly releasing the encapsulated contents in less than 5 s. The optimal AuNP packing in the wall, moderated by the custom ligands and verified using small angle x-ray spectroscopy, allows us to calculate the heat released in this process, and to simulate the temperature increase originating from the photothermal heating, with great accuracy. Atypically, we find the local heating does not cause a rise of more than 50 °C, which addresses a major shortcoming in plasmon actuated cargo delivery systems. This combination of spectral selectivity, low power requirements, low heat production, and fast release times, along with the versatility in terms of identity of the enclosed cargo, makes these hierarchical microshells suitable for wide-ranging applications, including biological ones.

  View details for PubMedID 29259223

• Dash-and-Recruit Mechanism Drives Membrane Curvature Recognition by the Small Bacterial Protein SpoVM CELL SYSTEMS Kim, E. Y., Tyndall, E. R., Huang, K., Tian, F., Ramamurthi, K. S. 2017; 5 (5): 518-+

  Abstract

  In Bacillus subtilis, sporulation requires that the 26-amino acid protein SpoVM embeds specifically into the forespore membrane, a structure with convex curvature. How this nanometer-sized protein can detect curves on a micrometer scale is not well understood. Here, we report that SpoVM exploits a "dash-and-recruit" mechanism to preferentially accumulate on the forespore. Using time-resolved imaging and flow cytometry, we observe that SpoVM exhibits a faster adsorption rate onto membranes of higher convex curvature. This preferential adsorption is accurately modeled as a two-step process: first, an initial binding event occurs with a faster on rate, then cooperative recruitment of additional SpoVM molecules follows. We demonstrate that both this biochemical process and effective sporulation in vivo require an unstructured and flexible SpoVM N terminus. We propose that this two-pronged strategy of fast adsorption followed by recruitment of subsequent molecules is a general mechanism that allows small proteins to detect subtle curves with a radius 1,000-fold their size.

  View details for PubMedID 29102609

• Deep Phenotypic Mapping of Bacterial Cytoskeletal Mutants Reveals Physiological Robustness to Cell Size CURRENT BIOLOGY Shi, H., Colavin, A., Bigos, M., Tropini, C., Monds, R. D., Huang, K. 2017; 27 (22): 3419-+

  Abstract

  Size is a universally defining characteristic of all living cells and tissues and is intrinsically linked with cell genotype, growth, and physiology. Many mutations have been identified to alter cell size, but pleiotropic effects have largely hampered our ability to probe how cell size specifically affects fundamental cellular properties, such as DNA content and intracellular localization. To systematically interrogate the impact of cell morphology on bacterial physiology, we used fluorescence-activated cell sorting to enrich a library of hundreds of Escherichia coli mutants in the essential cytoskeletal protein MreB for subtle changes in cell shape, cumulatively spanning ∼5-fold variation in average cell volume. Critically, pleiotropic effects in the mutated library are most likely minimized because only one gene was mutated and because growth rate was unaffected, thereby allowing us to query the general effects of morphology on cellular physiology over a large range of cell sizes with high resolution. We discovered linear scaling of the abundance of DNA and the key division protein FtsZ with cell volume, a strong dependency of sensitivity to specific antibiotics on cell width, and a simple correlation between MreB localization pattern and cell width. Our systematic, quantitative approach reveals complex and dynamic links between bacterial morphology and physiology and should be generally applicable for probing size-related genotype-phenotype relationships.

  View details for PubMedID 29103935

• Sizing up the bacterial cell cycle. Nature reviews. Microbiology Willis, L., Huang, K. C. 2017; 15 (10): 606-620

  Abstract

  It is remarkable how robustly a bacterial species can maintain its preferred size. This capacity is intimately related to control of the cell cycle: cell size and growth rate determine the duration of the cell cycle, which must accommodate the initiation and completion of DNA replication, and the assembly of the division apparatus during steady growth. Although we still lack an integrated view of the interconnections among events in the cell cycle, cell growth and cell size, the development of high-throughput imaging and image-processing protocols has stimulated a renaissance in the field. In this Review, we summarize recent findings, present simple classic models for cell size control, introduce high-throughput data-collection techniques, and explore the mechanisms that coordinate cell size with essential growth and cell cycle processes.

  View details for DOI 10.1038/nrmicro.2017.79

  View details for PubMedID 28804128

• Thinking big: the tunability of bacterial cell size FEMS MICROBIOLOGY REVIEWS Cesar, S., Huang, K. 2017; 41 (5): 672–78

  Abstract

  The determination of cell size is a fundamental challenge for all living organisms. In a given growth condition, cell size for a particular bacterial species typically falls within a narrow distribution. Nonetheless, size can vary enormously across species, and the size of a single bacterium can even vary substantially across growth conditions. Recent phenomenological studies have revived classic interest in how cells maintain their size and how they adjust their size with changes in growth rate. However, the mechanisms by which cells establish a particular size are relatively enigmatic. Here, we review existing knowledge on how size in rod-shaped bacteria is shaped by nutrient, mechanical, and genetic factors. We also examine obstacles to accurate size measurement and recent technologies that help to overcome these hurdles. Finally, we discuss the relevance of cell size to bacterial physiology.

  View details for PubMedID 28961755

  View details for PubMedCentralID PMC5812501

• Full color palette of fluorescent D-amino acids for in situ labeling of bacterial cell walls CHEMICAL SCIENCE Hsu, Y., Rittichier, J., Kuru, E., Yablonowski, J., Pasciak, E., Tekkam, S., Hall, E., Murphy, B., Lee, T. K., Garner, E. C., Huang, K., Brun, Y. V., VanNieuwenhze, M. S. 2017; 8 (9): 6313–21

  Abstract

  Fluorescent d-amino acids (FDAAs) enable efficient in situ labeling of peptidoglycan in diverse bacterial species. Conducted by enzymes involved in peptidoglycan biosynthesis, FDAA labeling allows specific probing of cell wall formation/remodeling activity, bacterial growth and cell morphology. Their broad application and high biocompatibility have made FDAAs an important and effective tool for studies of peptidoglycan synthesis and dynamics, which, in turn, has created a demand for the development of new FDAA probes. Here, we report the synthesis of new FDAAs, with emission wavelengths that span the entire visible spectrum. We also provide data to characterize their photochemical and physical properties, and we demonstrate their utility for visualizing peptidoglycan synthesis in Gram-negative and Gram-positive bacterial species. Finally, we show the permeability of FDAAs toward the outer-membrane of Gram-negative organisms, pinpointing the probes available for effective labeling in these species. This improved FDAA toolkit will enable numerous applications for the study of peptidoglycan biosynthesis and dynamics.

  View details for PubMedID 28989665

  View details for PubMedCentralID PMC5628581

• Cell Size: Fat Makes Cells Fat. Current biology : CB Willis, L., Huang, K. C. 2017; 27 (12): R592-R594

  Abstract

  Nutrients are required for the multiple biosynthetic pathways that result in cell growth, and faster growth due to increased nutrient supply results in larger cell volume. A new study demonstrates that fatty-acid availability limits growth rate and cell envelope capacity, revealing that fatty-acid synthesis is the primary determinant of cell size in bacteria and in budding yeast.

  View details for DOI 10.1016/j.cub.2017.05.017

  View details for PubMedID 28633027

• The Gut Microbiome: Connecting Spatial Organization to Function CELL HOST & MICROBE Tropini, C., Earle, K. A., Huang, K. C., Sonnenburg, J. L. 2017; 21 (4): 433-442

  Abstract

  The first rudimentary evidence that the human body harbors a microbiota hinted at the complexity of host-associated microbial ecosystems. Now, almost 400 years later, a renaissance in the study of microbiota spatial organization, driven by coincident revolutions in imaging and sequencing technologies, is revealing functional relationships between biogeography and health, particularly in the vertebrate gut. In this Review, we present our current understanding of principles governing the localization of intestinal bacteria, and spatial relationships between bacteria and their hosts. We further discuss important emerging directions that will enable progressing from the inherently descriptive nature of localization and -omics technologies to provide functional, quantitative, and mechanistic insight into this complex ecosystem.

  View details for DOI 10.1016/j.chom.2017.03.010

  View details for Web of Science ID 000398896100005

  View details for PubMedID 28407481

• Coupling between Protein Stability and Catalytic Activity Determines Pathogenicity of G6PD Variants CELL REPORTS Cunningham, A. D., Colavin, A., Huang, K. C., Mochly-Rosen, D. 2017; 18 (11): 2592-2599

  Abstract

  G6PD deficiency, an enzymopathy affecting 7% of the world population, is caused by over 160 identified amino acid variants in glucose-6-phosphate dehydrogenase (G6PD). The clinical presentation of G6PD deficiency is diverse, likely due to the broad distribution of variants across the protein and the potential for multidimensional biochemical effects. In this study, we use bioinformatic and biochemical analyses to interpret the relationship between G6PD variants and their clinical phenotype. Using structural information and statistical analyses of known G6PD variants, we predict the molecular phenotype of five uncharacterized variants from a reference population database. Through multidimensional analysis of biochemical data, we demonstrate that the clinical phenotypes of G6PD variants are largely determined by a trade-off between protein stability and catalytic activity. This work expands the current understanding of the biochemical underpinnings of G6PD variant pathogenicity and suggests a promising avenue for correcting G6PD deficiency by targeting essential structural features of G6PD.

  View details for DOI 10.1016/j.celrep.2017.02.048

  View details for PubMedID 28297664

• Emergent Phototactic Responses of Cyanobacteria under Complex Light Regimes MBIO Chau, R. M., Bhaya, D., Huang, K. C. 2017; 8 (2)

  Abstract

  Environmental cues can stimulate a variety of single-cell responses, as well as collective behaviors that emerge within a bacterial community. These responses require signal integration and transduction, which can occur on a variety of time scales and often involve feedback between processes, for example, between growth and motility. Here, we investigate the dynamics of responses of the phototactic, unicellular cyanobacterium Synechocystis sp. PCC6803 to complex light inputs that simulate the natural environments that cells typically encounter. We quantified single-cell motility characteristics in response to light of different wavelengths and intensities. We found that red and green light primarily affected motility bias rather than speed, while blue light inhibited motility altogether. When light signals were simultaneously presented from different directions, cells exhibited phototaxis along the vector sum of the light directions, indicating that cells can sense and combine multiple signals into an integrated motility response. Under a combination of antagonistic light signal regimes (phototaxis-promoting green light and phototaxis-inhibiting blue light), the ensuing bias was continuously tuned by competition between the wavelengths, and the community response was dependent on both bias and cell growth. The phototactic dynamics upon a rapid light shift revealed a wavelength dependence on the time scales of photoreceptor activation/deactivation. Thus, Synechocystis cells achieve exquisite integration of light inputs at the cellular scale through continuous tuning of motility, and the pattern of collective behavior depends on single-cell motility and population growth.IMPORTANCE The photosynthetic cyanobacterium Synechocystis sp. exhibits phototaxis that is dependent on the incident light wavelength through the action of various photoreceptors. In natural environments, cells experience a set of highly dynamic and complex light inputs, yet how cells transduce multiple or dynamic inputs into motion is unknown. In this study, we measured the phototactic behaviors of single cells and communities as a function of light intensity or when illuminated by combinations of lights of different wavelengths or incidence directions. Responses to a spectrum of light regimes revealed that Synechocystis sp. integrates information about the light environment to tune its phototactic response, which is likely generated by competition among photoreceptors and the degree of wavelength-regulated growth to sensitively control the direction and degree of movement.

  View details for DOI 10.1128/mBio.02330-16

  View details for Web of Science ID 000400575700011

  View details for PubMedID 28270586

• Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library. BMC biology Ursell, T., Lee, T. K., Shiomi, D., Shi, H., Tropini, C., Monds, R. D., Colavin, A., Billings, G., Bhaya-Grossman, I., Broxton, M., Huang, B. E., Niki, H., Huang, K. C. 2017; 15 (1): 17-?

  Abstract

  The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features.Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics.Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

  View details for DOI 10.1186/s12915-017-0348-8

  View details for PubMedID 28222723

  View details for PubMedCentralID PMC5320674

• GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science Yang, X., Lyu, Z., Miguel, A., McQuillen, R., Huang, K. C., Xiao, J. 2017; 355 (6326): 744-747

  Abstract

  The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.

  View details for DOI 10.1126/science.aak9995

  View details for PubMedID 28209899

• Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing BMC BIOLOGY Yu, F. B., Willis, L., Chau, R. M., Zambon, A., Horowitz, M., Bhaya, D., Huang, K. C., Quake, S. R. 2017; 15

  Abstract

  Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control.Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.

  View details for DOI 10.1186/s12915-016-0344-4

  View details for Web of Science ID 000394057800001

  View details for PubMedID 28196492

• Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates. Nature protocols Shi, H., Colavin, A., Lee, T. K., Huang, K. C. 2017; 12 (2): 429-438

  Abstract

  Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

  View details for DOI 10.1038/nprot.2016.181

  View details for PubMedID 28125106

• A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis. Cell Bartlett, T. M., Bratton, B. P., Duvshani, A., Miguel, A., Sheng, Y., Martin, N. R., Nguyen, J. P., Persat, A., Desmarais, S. M., VanNieuwenhze, M. S., Huang, K. C., Zhu, J., Shaevitz, J. W., Gitai, Z. 2017; 168 (1-2): 172-185 e15

  Abstract

  Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

  View details for DOI 10.1016/j.cell.2016.12.019

  View details for PubMedID 28086090

  View details for PubMedCentralID PMC5287421

• Staying in Touch while on the Go. Cell Huang, K. C. 2017; 168 (1-2): 15-17

  Abstract

  While chemical forms of cell-to-cell communication are well recognized to coordinate bacterial populations, electrical signaling has been relatively ignored. Humphries et al. show that Bacillus subtilis biofilms utilize potassium production to attract far away, motile cells of even phylogenetically distant species by altering their membrane potential.

  View details for DOI 10.1016/j.cell.2016.12.024

  View details for PubMedID 28086088

• Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche. Proceedings of the National Academy of Sciences of the United States of America Willis, L., Refahi, Y., Wightman, R., Landrein, B., Teles, J., Huang, K. C., Meyerowitz, E. M., Jönsson, H. 2016; 113 (51): E8238-E8246

  Abstract

  Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3-4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell-cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control.

  View details for DOI 10.1073/pnas.1616768113

  View details for PubMedID 27930326

• Single-molecule imaging reveals modulation of cell wall synthesis dynamics in live bacterial cells NATURE COMMUNICATIONS Lee, T. K., Meng, K., Shi, H., Huang, K. C. 2016; 7

  Abstract

  The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.

  View details for DOI 10.1038/ncomms13170

  View details for Web of Science ID 000385916600001

  View details for PubMedID 27774981

  View details for PubMedCentralID PMC5078992

• FtsZ-Dependent Elongation of a Coccoid Bacterium MBIO Pereira, A. R., Hsin, J., Krol, E., Tavares, A. C., Flores, P., Hoiczyk, E., Ng, N., Dajkovic, A., Brun, Y. V., VanNieuwenhze, M. S., Roemer, T., Carballido-Lopez, R., Scheffers, D., Huang, K. C., Pinho, M. G. 2016; 7 (5)

  Abstract

  A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ(G193D) allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ(G193D) filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci.The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

  View details for DOI 10.1128/mBio.00908-16

  View details for Web of Science ID 000390132900054

  View details for PubMedID 27601570

  View details for PubMedCentralID PMC5013293

• Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness. Cell systems Auer, G. K., Lee, T. K., Rajendram, M., Cesar, S., Miguel, A., Huang, K. C., Weibel, D. B. 2016; 2 (6): 402-411

  Abstract

  Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

  View details for DOI 10.1016/j.cels.2016.05.006

  View details for PubMedID 27321372

• A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria CELL Peters, J. M., Colavin, A., Shi, H., Czarny, T. L., Larson, M. H., Wong, S., Hawkins, J. S., Lu, C. H., Koo, B., Marta, E., Shiver, A. L., Whitehead, E. H., Weissman, J. S., Brown, E. D., Qi, L. S., Huang, K. C., Gross, C. A. 2016; 165 (6): 1493-1506

  Abstract

  Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

  View details for DOI 10.1016/j.cell.2016.05.003

  View details for Web of Science ID 000377045400021

  View details for PubMedID 27238023

  View details for PubMedCentralID PMC4894308

• The effect of microbial colonization on the host proteome varies by gastrointestinal location ISME JOURNAL Lichtman, J. S., Alsentzer, E., Jaffe, M., Sprockett, D., Masutani, E., Ikwa, E., Fragiadakis, G. K., Clifford, D., Huang, B. E., Sonnenburg, J. L., Huang, K. C., Elias, J. E. 2016; 10 (5): 1170-1181

  Abstract

  Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.

  View details for DOI 10.1038/ismej.2015.187

  View details for Web of Science ID 000374377200014

  View details for PubMedID 26574685

  View details for PubMedCentralID PMC5029216

• Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sutterlin, H. A., Shi, H., May, K. L., Miguel, A., Khare, S., Huang, K. C., Silhavy, T. J. 2016; 113 (11): E1565-E1574

  View details for DOI 10.1073/pnas.1601375113

  View details for Web of Science ID 000372014200020

• Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway. Proceedings of the National Academy of Sciences of the United States of America Sutterlin, H. A., Shi, H., May, K. L., Miguel, A., Khare, S., Huang, K. C., Silhavy, T. J. 2016; 113 (11): E1565-74

  Abstract

  Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.

  View details for DOI 10.1073/pnas.1601375113

  View details for PubMedID 26929379

  View details for PubMedCentralID PMC4801249

• High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography JOURNAL OF BIOLOGICAL CHEMISTRY Desmarais, S. M., Tropini, C., Miguel, A., Cava, F., Monds, R. D., de Pedro, M. A., Huang, K. C. 2015; 290 (52): 31090-31100

  View details for DOI 10.1074/jbc.M115.661660

  View details for PubMedID 26468288

• Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics BIOPHYSICAL JOURNAL Ando, D., Korabel, N., Huang, K. C., Gopinathan, A. 2015; 109 (8): 1574-1582

  View details for DOI 10.1016/j.bpj.2015.08.034

  View details for PubMedID 26488648

• Quantitative Imaging of Gut Microbiota Spatial Organization CELL HOST & MICROBE Earle, K. A., Billings, G., Sigal, M., Lichtman, J. S., Hansson, G. C., Elias, J. E., Amieva, M. R., Huang, K. C., Sonnenburg, J. L. 2015; 18 (4): 478-488

  Abstract

  Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.

  View details for DOI 10.1016/j.chom.2015.09.002

  View details for Web of Science ID 000365111600016

  View details for PubMedID 26439864

• Applications of imaging for bacterial systems biology CURRENT OPINION IN MICROBIOLOGY Huang, K. C. 2015; 27: 114-120

  View details for DOI 10.1016/j.mib.2015.08.003

  View details for PubMedID 26356259

• How Does the Xenopus laevis Embryonic Cell Cycle Avoid Spatial Chaos? CELL REPORTS Gelens, L., Huang, K. C., Ferrell, J. E. 2015; 12 (5): 892-900

  Abstract

  Theoretical studies have shown that a deterministic biochemical oscillator can become chaotic when operating over a sufficiently large volume and have suggested that the Xenopus laevis cell cycle oscillator operates close to such a chaotic regime. To experimentally test this hypothesis, we decreased the speed of the post-fertilization calcium wave, which had been predicted to generate chaos. However, cell divisions were found to develop normally, and eggs developed into normal tadpoles. Motivated by these experiments, we carried out modeling studies to understand the prerequisites for the predicted spatial chaos. We showed that this type of spatial chaos requires oscillatory reaction dynamics with short pulse duration and postulated that the mitotic exit in Xenopus laevis is likely slow enough to avoid chaos. In systems with shorter pulses, chaos may be an important hazard, as in cardiac arrhythmias, or a useful feature, as in the pigmentation of certain mollusk shells.

  View details for DOI 10.1016/j.celrep.2015.06.070

  View details for PubMedID 26212326

• Nanoengineering: Super symmetry in cell division. Nature nanotechnology Huang, K. C. 2015; 10 (8): 655-6

  View details for DOI 10.1038/nnano.2015.148

  View details for PubMedID 26242999

• The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction NATURE COMMUNICATIONS Sundararajan, K., Miguel, A., Desmarais, S. M., Meier, E. L., Huang, K. C., Goley, E. D. 2015; 6

  Abstract

  The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with β-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

  View details for DOI 10.1038/ncomms8281

  View details for Web of Science ID 000357170800008

  View details for PubMedCentralID PMC4532373

• Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division ELIFE Gray, A. N., Egan, A. J., Van't Veer, I. L., Verheul, J., Colavin, A., Koumoutsi, A., Biboy, J., Altelaar, A. F., Damen, M. J., Huang, K. C., Simorre, J., Breukink, E., den Blaauwen, T., Typas, A., Gross, C. A., Vollmer, W. 2015; 4

  Abstract

  To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.

  View details for DOI 10.7554/eLife.07118

  View details for Web of Science ID 000356145700001

  View details for PubMedID 25951518

  View details for PubMedCentralID PMC4458516

• Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus SCIENCE Zhou, X., Halladin, D. K., Rojas, E. R., Koslover, E. F., Lee, T. K., Huang, K. C., Theriot, J. A. 2015; 348 (6234): 574-578

  Abstract

  When Staphylococcus aureus undergoes cytokinesis, it builds a septum, generating two hemispherical daughters whose cell walls are only connected via a narrow peripheral ring. We found that resolution of this ring occurred within milliseconds ("popping"), without detectable changes in cell volume. The likelihood of popping depended on cell-wall stress, and the separating cells split open asymmetrically, leaving the daughters connected by a hinge. An elastostatic model of the wall indicated high circumferential stress in the peripheral ring before popping. Last, we observed small perforations in the peripheral ring that are likely initial points of mechanical failure. Thus, the ultrafast daughter cell separation in S. aureus appears to be driven by accumulation of stress in the peripheral ring and exhibits hallmarks of mechanical crack propagation.

  View details for DOI 10.1126/science.aaa1511

  View details for Web of Science ID 000353778100042

• Bacterial division. Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus. Science Zhou, X., Halladin, D. K., Rojas, E. R., Koslover, E. F., Lee, T. K., Huang, K. C., Theriot, J. A. 2015; 348 (6234): 574-578

  Abstract

  When Staphylococcus aureus undergoes cytokinesis, it builds a septum, generating two hemispherical daughters whose cell walls are only connected via a narrow peripheral ring. We found that resolution of this ring occurred within milliseconds ("popping"), without detectable changes in cell volume. The likelihood of popping depended on cell-wall stress, and the separating cells split open asymmetrically, leaving the daughters connected by a hinge. An elastostatic model of the wall indicated high circumferential stress in the peripheral ring before popping. Last, we observed small perforations in the peripheral ring that are likely initial points of mechanical failure. Thus, the ultrafast daughter cell separation in S. aureus appears to be driven by accumulation of stress in the peripheral ring and exhibits hallmarks of mechanical crack propagation.

  View details for DOI 10.1126/science.aaa1511

  View details for PubMedID 25931560

• Structural basis for the geometry-driven localization of a small protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gill, R. L., Castaing, J., Hsin, J., Tan, I. S., Wang, X., Huang, K. C., Tian, F., Ramamurthi, K. S. 2015; 112 (15): E1908-E1915

  Abstract

  In bacteria, certain shape-sensing proteins localize to differently curved membranes. During sporulation in Bacillus subtilis, the only convex (positively curved) surface in the cell is the forespore, an approximately spherical internal organelle. Previously, we demonstrated that SpoVM localizes to the forespore by preferentially adsorbing onto slightly convex membranes. Here, we used NMR and molecular dynamics simulations of SpoVM and a localization mutant (SpoVM(P9A)) to reveal that SpoVM's atypical amphipathic α-helix inserts deeply into the membrane and interacts extensively with acyl chains to sense packing differences in differently curved membranes. Based on binding to spherical supported lipid bilayers and Monte Carlo simulations, we hypothesize that SpoVM's membrane insertion, along with potential cooperative interactions with other SpoVM molecules in the lipid bilayer, drives its preferential localization onto slightly convex membranes. Such a mechanism, which is distinct from that used by high curvature-sensing proteins, may be widely conserved for the localization of proteins onto the surface of cellular organelles.

  View details for DOI 10.1073/pnas.1423868112

  View details for Web of Science ID 000352856800015

  View details for PubMedID 25825747

  View details for PubMedCentralID PMC4403194

• Maintenance of Motility Bias during Cyanobacterial Phototaxis BIOPHYSICAL JOURNAL Chau, R. M., Ursell, T., Wang, S., Huang, K. C., Bhaya, D. 2015; 108 (7): 1623-1632

  Abstract

  Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.

  View details for DOI 10.1016/j.bpj.2015.01.042

  View details for Web of Science ID 000352498100010

  View details for PubMedID 25863054

  View details for PubMedCentralID PMC4390813

• Variations in the binding pocket of an inhibitor of the bacterial division protein FtsZ across genotypes and species. PLoS computational biology Miguel, A., Hsin, J., Liu, T., Tang, G., Altman, R. B., Huang, K. C. 2015; 11 (3)

  Abstract

  The recent increase in antibiotic resistance in pathogenic bacteria calls for new approaches to drug-target selection and drug development. Targeting the mechanisms of action of proteins involved in bacterial cell division bypasses problems associated with increasingly ineffective variants of older antibiotics; to this end, the essential bacterial cytoskeletal protein FtsZ is a promising target. Recent work on its allosteric inhibitor, PC190723, revealed in vitro activity on Staphylococcus aureus FtsZ and in vivo antimicrobial activities. However, the mechanism of drug action and its effect on FtsZ in other bacterial species are unclear. Here, we examine the structural environment of the PC190723 binding pocket using PocketFEATURE, a statistical method that scores the similarity between pairs of small-molecule binding sites based on 3D structure information about the local microenvironment, and molecular dynamics (MD) simulations. We observed that species and nucleotide-binding state have significant impacts on the structural properties of the binding site, with substantially disparate microenvironments for bacterial species not from the Staphylococcus genus. Based on PocketFEATURE analysis of MD simulations of S. aureus FtsZ bound to GTP or with mutations that are known to confer PC190723 resistance, we predict that PC190723 strongly prefers to bind Staphylococcus FtsZ in the nucleotide-bound state. Furthermore, MD simulations of an FtsZ dimer indicated that polymerization may enhance PC190723 binding. Taken together, our results demonstrate that a drug-binding pocket can vary significantly across species, genetic perturbations, and in different polymerization states, yielding important information for the further development of FtsZ inhibitors.

  View details for DOI 10.1371/journal.pcbi.1004117

  View details for PubMedID 25811761

  View details for PubMedCentralID PMC4374959

• Variations in the Binding Pocket of an Inhibitor of the Bacterial Division Protein FtsZ across Genotypes and Species PLOS COMPUTATIONAL BIOLOGY Miguel, A., Hsin, J., Liu, T., Tang, G., Altman, R. B., Huang, K. C. 2015; 11 (3)

  Abstract

  The recent increase in antibiotic resistance in pathogenic bacteria calls for new approaches to drug-target selection and drug development. Targeting the mechanisms of action of proteins involved in bacterial cell division bypasses problems associated with increasingly ineffective variants of older antibiotics; to this end, the essential bacterial cytoskeletal protein FtsZ is a promising target. Recent work on its allosteric inhibitor, PC190723, revealed in vitro activity on Staphylococcus aureus FtsZ and in vivo antimicrobial activities. However, the mechanism of drug action and its effect on FtsZ in other bacterial species are unclear. Here, we examine the structural environment of the PC190723 binding pocket using PocketFEATURE, a statistical method that scores the similarity between pairs of small-molecule binding sites based on 3D structure information about the local microenvironment, and molecular dynamics (MD) simulations. We observed that species and nucleotide-binding state have significant impacts on the structural properties of the binding site, with substantially disparate microenvironments for bacterial species not from the Staphylococcus genus. Based on PocketFEATURE analysis of MD simulations of S. aureus FtsZ bound to GTP or with mutations that are known to confer PC190723 resistance, we predict that PC190723 strongly prefers to bind Staphylococcus FtsZ in the nucleotide-bound state. Furthermore, MD simulations of an FtsZ dimer indicated that polymerization may enhance PC190723 binding. Taken together, our results demonstrate that a drug-binding pocket can vary significantly across species, genetic perturbations, and in different polymerization states, yielding important information for the further development of FtsZ inhibitors.

  View details for DOI 10.1371/journal.pcbi.1004117

  View details for Web of Science ID 000352195700026

  View details for PubMedID 25811761

  View details for PubMedCentralID PMC4374959

• The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis. Molecular biology of the cell Zhou, Z., Munteanu, E. L., He, J., Ursell, T., Bathe, M., Huang, K. C., Chang, F. 2015; 26 (1): 78-90

  Abstract

  The functions of the actin-myosin-based contractile ring in cytokinesis remain to be elucidated. Recent findings show that in the fission yeast Schizosaccharomyces pombe, cleavage furrow ingression is driven by polymerization of cell wall fibers outside the plasma membrane, not by the contractile ring. Here we show that one function of the ring is to spatially coordinate septum cell wall assembly. We develop an improved method for live-cell imaging of the division apparatus by orienting the rod-shaped cells vertically using microfabricated wells. We observe that the septum hole and ring are circular and centered in wild-type cells and that in the absence of a functional ring, the septum continues to ingress but in a disorganized and asymmetric manner. By manipulating the cleavage furrow into different shapes, we show that the ring promotes local septum growth in a curvature-dependent manner, allowing even a misshapen septum to grow into a more regular shape. This curvature-dependent growth suggests a model in which contractile forces of the ring shape the septum cell wall by stimulating the cell wall machinery in a mechanosensitive manner. Mechanical regulation of the cell wall assembly may have general relevance to the morphogenesis of walled cells.

  View details for DOI 10.1091/mbc.E14-10-1441

  View details for PubMedID 25355954

  View details for PubMedCentralID PMC4279231

• Physics of Intracellular Organization in Bacteria ANNUAL REVIEW OF MICROBIOLOGY, VOL 69 Wingreen, N. S., Huang, K. C. 2015; 69: 361-379

  View details for DOI 10.1146/annurev-micro-091014-104313

  View details for Web of Science ID 000363614100019

  View details for PubMedID 26488278

• Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations CELL REPORTS Tropini, C., Lee, T. K., Hsin, J., Desmarais, S. M., Ursell, T., Monds, R. D., Huang, K. C. 2014; 9 (4): 1520-1527

  Abstract

  Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  View details for DOI 10.1016/j.celrep.2014.10.027

  View details for Web of Science ID 000345529600031

  View details for PubMedCentralID PMC4254626

• Principles of bacterial cell-size determination revealed by cell-wall synthesis perturbations. Cell reports Tropini, C., Lee, T. K., Hsin, J., Desmarais, S. M., Ursell, T., Monds, R. D., Huang, K. C. 2014; 9 (4): 1520-1527

  Abstract

  Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  View details for DOI 10.1016/j.celrep.2014.10.027

  View details for PubMedID 25456140

• Systematic perturbation of cytoskeletal function reveals a linear scaling relationship between cell geometry and fitness. Cell reports Monds, R. D., Lee, T. K., Colavin, A., Ursell, T., Quan, S., Cooper, T. F., Huang, K. C. 2014; 9 (4): 1528-1537

  Abstract

  Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  View details for DOI 10.1016/j.celrep.2014.10.040

  View details for PubMedID 25456141

• Systematic Perturbation of Cytoskeletal Function Reveals a Linear Scaling Relationship between Cell Geometry and Fitness CELL REPORTS Monds, R. D., Lee, T. K., Colavin, A., Ursell, T., Quan, S., Cooper, T. F., Huang, K. C. 2014; 9 (4): 1528-1537

  Abstract

  Diversification of cell size is hypothesized to have occurred through a process of evolutionary optimization, but direct demonstrations of causal relationships between cell geometry and fitness are lacking. Here, we identify a mutation from a laboratory-evolved bacterium that dramatically increases cell size through cytoskeletal perturbation and confers a large fitness advantage. We engineer a library of cytoskeletal mutants of different sizes and show that fitness scales linearly with respect to cell size over a wide physiological range. Quantification of the growth rates of single cells during the exit from stationary phase reveals that transitions between "feast-or-famine" growth regimes are a key determinant of cell-size-dependent fitness effects. We also uncover environments that suppress the fitness advantage of larger cells, indicating that cell-size-dependent fitness effects are subject to both biophysical and metabolic constraints. Together, our results highlight laboratory-based evolution as a powerful framework for studying the quantitative relationships between morphology and fitness.

  View details for DOI 10.1016/j.celrep.2014.10.040

  View details for Web of Science ID 000345529600032

• De novo morphogenesis in L-forms via geometric control of cell growth. Molecular microbiology Billings, G., Ouzounov, N., Ursell, T., Desmarais, S. M., Shaevitz, J., Gitai, Z., Huang, K. C. 2014; 93 (5): 883-896

  Abstract

  In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod-like shape is maintained via the spatiotemporal patterning of cell-wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell-wall synthesis in E. coli to generate cell-wall-deficient, spherical L-forms, and found that they robustly reverted to a rod-like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod-like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.

  View details for DOI 10.1111/mmi.12703

  View details for PubMedID 24995493

• How and why cells grow as rods BMC BIOLOGY Chang, F., Huang, K. C. 2014; 12

  View details for DOI 10.1186/s12915-014-0054-8

  View details for Web of Science ID 000339839500001

• Response of Escherichia coli growth rate to osmotic shock PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rojas, E., Theriot, J. A., Huang, K. C. 2014; 111 (21): 7807-7812

  Abstract

  It has long been proposed that turgor pressure plays an essential role during bacterial growth by driving mechanical expansion of the cell wall. This hypothesis is based on analogy to plant cells, for which this mechanism has been established, and on experiments in which the growth rate of bacterial cultures was observed to decrease as the osmolarity of the growth medium was increased. To distinguish the effect of turgor pressure from pressure-independent effects that osmolarity might have on cell growth, we monitored the elongation of single Escherichia coli cells while rapidly changing the osmolarity of their media. By plasmolyzing cells, we found that cell-wall elastic strain did not scale with growth rate, suggesting that pressure does not drive cell-wall expansion. Furthermore, in response to hyper- and hypoosmotic shock, E. coli cells resumed their preshock growth rate and relaxed to their steady-state rate after several minutes, demonstrating that osmolarity modulates growth rate slowly, independently of pressure. Oscillatory hyperosmotic shock revealed that although plasmolysis slowed cell elongation, the cells nevertheless "stored" growth such that once turgor was reestablished the cells elongated to the length that they would have attained had they never been plasmolyzed. Finally, MreB dynamics were unaffected by osmotic shock. These results reveal the simple nature of E. coli cell-wall expansion: that the rate of expansion is determined by the rate of peptidoglycan insertion and insertion is not directly dependent on turgor pressure, but that pressure does play a basic role whereby it enables full extension of recently inserted peptidoglycan.

  View details for DOI 10.1073/pnas.1402591111

  View details for Web of Science ID 000336411300068

  View details for PubMedID 24821776

  View details for PubMedCentralID PMC4040581

• A dynamically assembled cell wall synthesis machinery buffers cell growth. Proceedings of the National Academy of Sciences of the United States of America Lee, T. K., Tropini, C., Hsin, J., Desmarais, S. M., Ursell, T. S., Gong, E., Gitai, Z., Monds, R. D., Huang, K. C. 2014; 111 (12): 4554-4559

  Abstract

  Assembly of protein complexes is a key mechanism for achieving spatial and temporal coordination in processes involving many enzymes. Growth of rod-shaped bacteria is a well-studied example requiring such coordination; expansion of the cell wall is thought to involve coordination of the activity of synthetic enzymes with the cytoskeleton via a stable complex. Here, we use single-molecule tracking to demonstrate that the bacterial actin homolog MreB and the essential cell wall enzyme PBP2 move on timescales orders of magnitude apart, with drastically different characteristic motions. Our observations suggest that PBP2 interacts with the rest of the synthesis machinery through a dynamic cycle of transient association. Consistent with this model, growth is robust to large fluctuations in PBP2 abundance. In contrast to stable complex formation, dynamic association of PBP2 is less dependent on the function of other components of the synthesis machinery, and buffers spatially distributed growth against fluctuations in pathway component concentrations and the presence of defective components. Dynamic association could generally represent an efficient strategy for spatiotemporal coordination of protein activities, especially when excess concentrations of system components are inhibitory to the overall process or deleterious to the cell.

  View details for DOI 10.1073/pnas.1313826111

  View details for PubMedID 24550500

• Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization. Proceedings of the National Academy of Sciences of the United States of America Ursell, T. S., Nguyen, J., Monds, R. D., Colavin, A., Billings, G., Ouzounov, N., Gitai, Z., Shaevitz, J. W., Huang, K. C. 2014; 111 (11): E1025-34

  Abstract

  Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

  View details for DOI 10.1073/pnas.1317174111

  View details for PubMedID 24550515

  View details for PubMedCentralID PMC3964057

• Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ursell, T. S., Nguyen, J., Monds, R. D., Colavin, A., Billings, G., Ouzounov, N., Gitai, Z., Shaevitz, J. W., Huang, K. C. 2014; 111 (11): E1025-E1034

  View details for DOI 10.1073/pnas.1317174111

  View details for Web of Science ID 000333027900012

  View details for PubMedID 24550515

• Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB. Proceedings of the National Academy of Sciences of the United States of America Colavin, A., Hsin, J., Huang, K. C. 2014; 111 (9): 3585-3590

  Abstract

  The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

  View details for DOI 10.1073/pnas.1317061111

  View details for PubMedID 24550504

  View details for PubMedCentralID PMC3948266

• Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. Journal of visualized experiments : JoVE Desmarais, S. M., Cava, F., de Pedro, M. A., Huang, K. C. 2014

  Abstract

  The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

  View details for DOI 10.3791/51183

  View details for PubMedID 24457605

• The role of hydrolases in bacterial cell-wall growth. Current opinion in microbiology Lee, T. K., Huang, K. C. 2013; 16 (6): 760-766

  Abstract

  Although hydrolysis is known to be as important as synthesis in the growth and development of the bacterial cell wall, the coupling between these processes is not well understood. Bond cleavage can generate deleterious pores, but may also be required for the incorporation of new material and for the expansion of the wall, highlighting the importance of mechanical forces in interpreting the consequences of hydrolysis in models of growth. Critically, minimal essential subsets of hydrolases have now been identified in several model organisms, enabling the reduction of genetic complexity. Recent studies in Bacillus subtilis have provided evidence for both the presence and absence of coupling between synthesis and hydrolysis during sporulation and elongation, respectively. In this review, we discuss strategies for dissecting the relationship between synthesis and hydrolysis using time-lapse imaging, biophysical measurements of cell-wall architecture, and computational modeling.

  View details for DOI 10.1016/j.mib.2013.08.005

  View details for PubMedID 24035761

• Dimer Dynamics and Filament Organization of the Bacterial Cell Division Protein FtsA. Journal of molecular biology Hsin, J., Fu, R., Huang, K. C. 2013; 425 (22): 4415-4426

  Abstract

  FtsA is a bacterial actin homolog and one of the core proteins involved in cell division. While previous studies have demonstrated the capability of FtsA to polymerize, little is known about its polymerization state in vivo, or if polymerization is necessary for FtsA function. Given that one function of FtsA is to tether FtsZ filaments to the membrane, in vivo polymerization of FtsA imposes geometric constraints and requires a specific polymer curvature direction. Here we report a series of molecular dynamics simulations probing the structural dynamics of FtsA as a dimer and as a tetrameric single filament. We found that the FtsA polymer exhibits a preferred bending direction that would allow for its placement parallel to FtsZ polymers underneath the cytoplasmic membrane. We also identified key interfacial amino acids that mediate FtsA-FtsA interaction, and propose that some amino acids play more critical roles than others. We performed in silico mutagenesis on FtsA and demonstrated that while a moderate mutation at the polymerization interface does not significantly affect polymer properties such as bending direction and association strength, more drastic mutations change both features and could lead to non-functional FtsA.

  View details for DOI 10.1016/j.jmb.2013.07.016

  View details for PubMedID 23871894

• Motility Enhancement through Surface Modification Is Sufficient for Cyanobacterial Community Organization during Phototaxis. PLoS computational biology Ursell, T., Chau, R. M., Wisen, S., Bhaya, D., Huang, K. C. 2013; 9 (9)

  View details for DOI 10.1371/journal.pcbi.1003205

  View details for PubMedID 24039562

• FtsZ Protofilaments Use a Hinge-Opening Mechanism for Constrictive Force Generation SCIENCE Li, Y., Hsin, J., Zhao, L., Cheng, Y., Shang, W., Huang, K. C., Wang, H., Ye, S. 2013; 341 (6144): 392-395

  Abstract

  The essential bacterial protein FtsZ is a guanosine triphosphatase that self-assembles into a structure at the division site termed the "Z ring". During cytokinesis, the Z ring exerts a constrictive force on the membrane by using the chemical energy of guanosine triphosphate hydrolysis. However, the structural basis of this constriction remains unresolved. Here, we present the crystal structure of a guanosine diphosphate-bound Mycobacterium tuberculosis FtsZ protofilament, which exhibits a curved conformational state. The structure reveals a longitudinal interface that is important for function. The protofilament curvature highlights a hydrolysis-dependent conformational switch at the T3 loop that leads to longitudinal bending between subunits, which could generate sufficient force to drive cytokinesis.

  View details for DOI 10.1126/science.1239248

  View details for Web of Science ID 000322259200048

  View details for PubMedID 23888039

• Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly. Molecular microbiology Desmarais, S. M., de Pedro, M. A., Cava, F., Huang, K. C. 2013; 89 (1): 1-13

  Abstract

  The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell wall synthesis and cell growth. High-performance liquid chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques.

  View details for DOI 10.1111/mmi.12266

  View details for PubMedID 23679048

• Optimal Dynamics for Quality Control in Spatially Distributed Mitochondrial Networks PLOS COMPUTATIONAL BIOLOGY Patel, P. K., Shirihai, O., Huang, K. C. 2013; 9 (7)

  Abstract

  Recent imaging studies of mitochondrial dynamics have implicated a cycle of fusion, fission, and autophagy in the quality control of mitochondrial function by selectively increasing the membrane potential of some mitochondria at the expense of the turnover of others. This complex, dynamical system creates spatially distributed networks that are dependent on active transport along cytoskeletal networks and on protein import leading to biogenesis. To study the relative impacts of local interactions between neighboring mitochondria and their reorganization via transport, we have developed a spatiotemporal mathematical model encompassing all of these processes in which we focus on the dynamics of a health parameter meant to mimic the functional state of mitochondria. In agreement with previous models, we show that both autophagy and the generation of membrane potential asymmetry following a fusion/fission cycle are required for maintaining a healthy mitochondrial population. This health maintenance is affected by mitochondrial density and motility primarily through changes in the frequency of fusion events. Health is optimized when the selectivity thresholds for fusion and fission are matched, providing a mechanistic basis for the observed coupling of the two processes through the protein OPA1. We also demonstrate that the discreteness of the components exchanged during fusion is critical for quality control, and that the effects of limiting total amounts of autophagy and biogenesis have distinct consequences on health and population size, respectively. Taken together, our results show that several general principles emerge from the complexity of the quality control cycle that can be used to focus and interpret future experimental studies, and our modeling framework provides a road-map for deconstructing the functional importance of local interactions in communities of cells as well as organelles.

  View details for DOI 10.1371/journal.pcbi.1003108

  View details for Web of Science ID 000322320200003

  View details for PubMedID 23874166

  View details for PubMedCentralID PMC3708874

• Design of High-Specificity Nanocarriers by Exploiting Non-Equilibrium Effects in Cancer Cell Targeting PLOS ONE Tsekouras, K., Goncharenko, I., Colvin, M. E., Huang, K. C., Gopinathan, A. 2013; 8 (6)

  View details for DOI 10.1371/journal.pone.0065623

  View details for Web of Science ID 000321424400009

• Mechanical consequences of cell-wall turnover in the elongation of a gram-positive bacterium. Biophysical journal Misra, G., Rojas, E. R., Gopinathan, A., Huang, K. C. 2013; 104 (11): 2342-2352

  Abstract

  A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.

  View details for DOI 10.1016/j.bpj.2013.04.047

  View details for PubMedID 23746506

  View details for PubMedCentralID PMC3672878

• Motility enhancement through surface modification is sufficient for cyanobacterial community organization during phototaxis. PLoS computational biology Ursell, T., Chau, R. M., Wisen, S., Bhaya, D., Huang, K. C. 2013; 9 (9)

  Abstract

  The emergent behaviors of communities of genotypically identical cells cannot be easily predicted from the behaviors of individual cells. In many cases, it is thought that direct cell-cell communication plays a critical role in the transition from individual to community behaviors. In the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803, individual cells exhibit light-directed motility ("phototaxis") over surfaces, resulting in the emergence of dynamic spatial organization of multicellular communities. To probe this striking community behavior, we carried out time-lapse video microscopy coupled with quantitative analysis of single-cell dynamics under varying light conditions. These analyses suggest that cells secrete an extracellular substance that modifies the physical properties of the substrate, leading to enhanced motility and the ability for groups of cells to passively guide one another. We developed a biophysical model that demonstrates that this form of indirect, surface-based communication is sufficient to create distinct motile groups whose shape, velocity, and dynamics qualitatively match our experimental observations, even in the absence of direct cellular interactions or changes in single-cell behavior. Our computational analysis of the predicted community behavior, across a matrix of cellular concentrations and light biases, demonstrates that spatial patterning follows robust scaling laws and provides a useful resource for the generation of testable hypotheses regarding phototactic behavior. In addition, we predict that degradation of the surface modification may account for the secondary patterns occasionally observed after the initial formation of a community structure. Taken together, our modeling and experiments provide a framework to show that the emergent spatial organization of phototactic communities requires modification of the substrate, and this form of surface-based communication could provide insight into the behavior of a wide array of biological communities.

  View details for DOI 10.1371/journal.pcbi.1003205

  View details for PubMedID 24039562

  View details for PubMedCentralID PMC3763999

• The role of hydrolases in bacterial cell-wall growth CurrOpinMicrobiol  Lee, T. K., Huang, K. C. 2013; 16: xx-yy
• Multiple conformations of FtsZ protofilaments provide structural insight into mechanisms of bacterial cytokinesis Science Li, Y., Hsin, J., Zhao, L., Cheng, Y., Huang, K. C., Wang, H. W. 2013; 341: 392-395
• Biological Consequences and Advantages of Asymmetric Bacterial Growth ANNUAL REVIEW OF MICROBIOLOGY, VOL 67 Kysela, D. T., Brown, P. J., Huang, K. C., Brun, Y. V. 2013; 67: 417-435

  Abstract

  Asymmetries in cell growth and division occur in eukaryotes and prokaryotes alike. Even seemingly simple and morphologically symmetric cell division processes belie inherent underlying asymmetries in the composition of the resulting daughter cells. We consider the types of asymmetry that arise in various bacterial cell growth and division processes, which include both conditionally activated mechanisms and constitutive, hardwired aspects of bacterial life histories. Although asymmetry disposes some cells to the deleterious effects of aging, it may also benefit populations by efficiently purging accumulated damage and rejuvenating newborn cells. Asymmetries may also generate phenotypic variation required for successful exploitation of variable environments, even when extrinsic changes outpace the capacity of cells to sense and respond to challenges. We propose specific experimental approaches to further develop our understanding of the prevalence and the ultimate importance of asymmetric bacterial growth.

  View details for DOI 10.1146/annurev-micro-092412-155622

  View details for Web of Science ID 000326686400021

  View details for PubMedID 23808335

• Optimal Nanocarrier Design for Cancer Cell Targeting PloS One Tsekouras, K., Goncharenko, I., Colvin, M., Huang, K. C., Gopinathan, A. 2013; 8: e65623
• Physiological role of FtsA polymerization during bacterial cell division J MolBiol Hsin, J., Fu, R., Huang, K. C. 2013; 425: 4415-4426 
• The molecular origins of chiral growth in walled cells CURRENT OPINION IN MICROBIOLOGY Huang, K. C., Ehrhardt, D. W., Shaevitz, J. W. 2012; 15 (6): 707-714

  Abstract

  Cells from all kingdoms of life adopt a dizzying array of fascinating shapes that support cellular function. Amoeboid and spherical shapes represent perhaps the simplest of geometries that may minimize the level of growth control required for survival. Slightly more complex are rod-shaped cells, from microscopic bacteria to macroscopic plants, which require additional mechanisms to define a cell's longitudinal axis, width, and length. Recent evidence suggests that many rod-shaped, walled cells achieve elongated growth through chiral insertion of cell-wall material that may be coupled to a twisting of the cell body. Inspired by these observations, biophysical mechanisms for twisting growth have been proposed that link the mechanics of intracellular proteins to cell shape maintenance. In this review, we highlight experimental and theoretical work that connects molecular-scale organization and structure with the cellular-scale phenomena of rod-shaped growth.

  View details for DOI 10.1016/j.mib.2012.11.002

  View details for Web of Science ID 000313612200012

  View details for PubMedID 23194654

• Analysis of Surface Protein Expression Reveals the Growth Pattern of the Gram-Negative Outer Membrane PLOS COMPUTATIONAL BIOLOGY Ursell, T. S., Trepagnier, E. H., Huang, K. C., Theriot, J. A. 2012; 8 (9)

  Abstract

  The outer membrane (OM) of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ≈ 10(-2) µm(2) of OM material per two minutes per µm(2). Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM.

  View details for DOI 10.1371/journal.pcbi.1002680

  View details for Web of Science ID 000309510900017

  View details for PubMedID 23028278

  View details for PubMedCentralID PMC3459847

• Physical constraints on the establishment of intracellular spatial gradients in bacteria BMC BIOPHYSICS Tropini, C., Rabbani, N., Huang, K. C. 2012; 5

  Abstract

  Bacteria dynamically regulate their intricate intracellular organization involving proteins that facilitate cell division, motility, and numerous other processes. Consistent with this sophisticated organization, bacteria are able to create asymmetries and spatial gradients of proteins by localizing signaling pathway components. We use mathematical modeling to investigate the biochemical and physical constraints on the generation of intracellular gradients by the asymmetric localization of a source and a sink.We present a systematic computational analysis of the effects of other regulatory mechanisms, such as synthesis, degradation, saturation, and cell growth. We also demonstrate that gradients can be established in a variety of bacterial morphologies such as rods, crescents, spheres, branched and constricted cells.Taken together, these results suggest that gradients are a robust and potentially common mechanism for providing intracellular spatial cues.

  View details for DOI 10.1186/2046-1682-5-17

  View details for Web of Science ID 000311052500001

  View details for PubMedID 22931750

  View details for PubMedCentralID PMC3496868

• Interplay between the Localization and Kinetics of Phosphorylation in Flagellar Pole Development of the Bacterium Caulobacter crescentus PLOS COMPUTATIONAL BIOLOGY Tropini, C., Huang, K. C. 2012; 8 (8)

  Abstract

  Bacterial cells maintain sophisticated levels of intracellular organization that allow for signal amplification, response to stimuli, cell division, and many other critical processes. The mechanisms underlying localization and their contribution to fitness have been difficult to uncover, due to the often challenging task of creating mutants with systematically perturbed localization but normal enzymatic activity, and the lack of quantitative models through which to interpret subtle phenotypic changes. Focusing on the model bacterium Caulobacter crescentus, which generates two different types of daughter cells from an underlying asymmetric distribution of protein phosphorylation, we use mathematical modeling to investigate the contribution of the localization of histidine kinases to the establishment of cellular asymmetry and subsequent developmental outcomes. We use existing mutant phenotypes and fluorescence data to parameterize a reaction-diffusion model of the kinases PleC and DivJ and their cognate response regulator DivK. We then present a systematic computational analysis of the effects of changes in protein localization and abundance to determine whether PleC localization is required for correct developmental timing in Caulobacter. Our model predicts the developmental phenotypes of several localization mutants, and suggests that a novel strain with co-localization of PleC and DivJ could provide quantitative insight into the signaling threshold required for flagellar pole development. Our analysis indicates that normal development can be maintained through a wide range of localization phenotypes, and that developmental defects due to changes in PleC localization can be rescued by increased PleC expression. We also show that the system is remarkably robust to perturbation of the kinetic parameters, and while the localization of either PleC or DivJ is required for asymmetric development, the delocalization of one of these two components does not prevent flagellar pole development. We further find that allosteric regulation of PleC observed in vitro does not affect the predicted in vivo developmental phenotypes. Taken together, our model suggests that cells can tolerate perturbations to localization phenotypes, whose evolutionary origins may be connected with reducing protein expression or with decoupling pre- and post-division phenotypes.

  View details for DOI 10.1371/journal.pcbi.1002602

  View details for Web of Science ID 000308553500004

  View details for PubMedID 22876167

  View details for PubMedCentralID PMC3410866

• Posttranslational Acetylation of alpha-Tubulin Constrains Protofilament Number in Native Microtubules CURRENT BIOLOGY Cueva, J. G., Hsin, J., Huang, K. C., Goodman, M. B. 2012; 22 (12): 1066-1074

  Abstract

  Microtubules are built from linear polymers of α-β tubulin dimers (protofilaments) that form a tubular quinary structure. Microtubules assembled from purified tubulin in vitro contain between 10 and 16 protofilaments; however, such structural polymorphisms are not found in cells. This discrepancy implies that factors other than tubulin constrain microtubule protofilament number, but the nature of these constraints is unknown.Here, we show that acetylation of MEC-12 α-tubulin constrains protofilament number in C. elegans touch receptor neurons (TRNs). Whereas the sensory dendrite of wild-type TRNs is packed with a cross-linked bundle of long, 15-protofilament microtubules, mec-17;atat-2 mutants lacking α-tubulin acetyltransferase activity have short microtubules, rampant lattice defects, and variable protofilament number both between and within microtubules. All-atom molecular dynamics simulations suggest a model in which acetylation of lysine 40 promotes the formation of interprotofilament salt bridges, stabilizing lateral interactions between protofilaments and constraining quinary structure to produce stable, structurally uniform microtubules in vivo.Acetylation of α-tubulin is an essential constraint on protofilament number in vivo. We propose a structural model in which this posttranslational modification promotes the formation of lateral salt bridges that fine-tune the association between adjacent protofilaments and enable the formation of uniform microtubule populations in vivo.

  View details for DOI 10.1016/j.cub.2012.05.012

  View details for Web of Science ID 000305766900020

  View details for PubMedID 22658592

  View details for PubMedCentralID PMC3670109

• Nucleotide-dependent conformations of FtsZ dimers and force generation observed through molecular dynamics simulations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hsin, J., Gopinathan, A., Huang, K. C. 2012; 109 (24): 9432-9437

  Abstract

  The bacterial cytoskeletal protein FtsZ is a GTPase that is thought to provide mechanical constriction force via an unidentified mechanism. Purified FtsZ polymerizes into filaments with varying structures in vitro: while GTP-bound FtsZ assembles into straight or gently curved filaments, GDP-bound FtsZ forms highly curved filaments, prompting the hypothesis that a difference in the inherent curvature of FtsZ filaments provides mechanical force. However, no nucleotide-dependent structural transition of FtsZ monomers has been observed to support this force generation model. Here, we present a series of all-atom molecular dynamics simulations probing the effects of nucleotide binding on the structure of an FtsZ dimer. We found that the FtsZ-dimer structure is dependent on nucleotide-binding state. While a GTP-bound FtsZ dimer retained a firm monomer-monomer contact, a GDP-bound FtsZ dimer lost some of the monomer-monomer association, leading to a "hinge-opening" event that resulted in a more bent dimer, while leaving each monomer structure largely unaffected. We constructed models of FtsZ filaments and found that a GDP-FtsZ filament is much more curved than a GTP-FtsZ filament, with the degree of curvature matching prior experimental data. FtsZ dynamics were used to estimate the amount of force an FtsZ filament could exert when hydrolysis occurs (20-30 pN per monomer). This magnitude of force is sufficient to direct inward cell-wall growth during division, and to produce the observed degree of membrane pinching in liposomes. Taken together, our data provide molecular-scale insight on the origin of FtsZ-based constriction force, and the mechanism underlying prokaryotic cell division.

  View details for DOI 10.1073/pnas.1120761109

  View details for Web of Science ID 000305511300049

  View details for PubMedID 22647609

  View details for PubMedCentralID PMC3386107

• Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity MOLECULAR MICROBIOLOGY Tuson, H. H., Auer, G. K., Renner, L. D., Hasebe, M., Tropini, C., Salick, M., Crone, W. C., Gopinathan, A., Huang, K. C., Weibel, D. B. 2012; 84 (5): 874-891

  Abstract

  Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user-defined stiffness, measured the growth rate of individual cells and fit data to a thin-shell mechanical model to extract the effective longitudinal Young's modulus of the cell envelope of Escherichia coli (50-150 MPa), Bacillus subtilis (100-200 MPa) and Pseudomonas aeruginosa (100-200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour-intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22-induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Young's modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.

  View details for DOI 10.1111/j.1365-2958.2012.08063.x

  View details for Web of Science ID 000304301500007

  View details for PubMedID 22548341

• Helical insertion of peptidoglycan produces chiral ordering of the bacterial cell wall PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, S., Furchtgott, L., Huang, K. C., Shaevitz, J. W. 2012; 109 (10): E595-E604

  Abstract

  The regulation of cell shape is a common challenge faced by organisms across all biological kingdoms. In nearly all bacteria, cell shape is determined by the architecture of the peptidoglycan cell wall, a macromolecule consisting of glycan strands crosslinked by peptides. In addition to shape, cell growth must also maintain the wall structural integrity to prevent lysis due to large turgor pressures. Robustness can be accomplished by establishing a globally ordered cell-wall network, although how a bacterium generates and maintains peptidoglycan order on the micron scale using nanometer-sized proteins remains a mystery. Here, we demonstrate that left-handed chirality of the MreB cytoskeleton in the rod-shaped bacterium Escherichia coli gives rise to a global, right-handed chiral ordering of the cell wall. Local, MreB-guided insertion of material into the peptidoglycan network naturally orders the glycan strands and causes cells to twist left-handedly during elongational growth. Through comparison with the right-handed twisting of Bacillus subtilis cells, our work supports a common mechanism linking helical insertion and chiral cell-wall ordering in rod-shaped bacteria. These physical principles of cell growth link the molecular structure of the bacterial cytoskeleton, mechanisms of wall synthesis, and the coordination of cell-wall architecture.

  View details for DOI 10.1073/pnas.1117132109

  View details for Web of Science ID 000301117700005

  View details for PubMedID 22343529

  View details for PubMedCentralID PMC3309786

• Alpha Tubulin Acetylation Regulates Protofilament Number in Native Microtubules Curr Biol Cueva, J., Hsin, J., Huang, K. C., Goodman, M. 2012; 22: 1066-1074
• Conformational changes, diffusion and collective behavior in monomeric kinesin-based motility JOURNAL OF PHYSICS-CONDENSED MATTER Huang, K. C., Vega, C., Gopinathan, A. 2011; 23 (37)

  Abstract

  Molecular motors convert chemical energy into mechanical motion and power the transport of material within living cells; the motion of a motor is thought to be influenced by stochastic chemical state transitions of the molecule as well as intramolecular diffusion of one motor head seeking the next binding site. Existing models for the motility of single-headed monomeric motors that map the system to a simplified two-state Brownian ratchet have some predictive power, but in general are unable to elucidate the contributions of different molecular level processes to the overall effective parameters. In this work, we build a detailed molecular level model of monomeric kinesin motility that naturally incorporates conformational changes (power strokes) and biased diffusion. Our results predict that mean velocity is most sensitive to the power stroke size, while run length distribution is sensitive primarily to the strength of the microtubule bias potential with a weak dependence on power stroke that can be tuned by the strength of an applied load. In addition, we demonstrate that motor pairs attached to the same cargo can cooperatively function to increase motility in both the plus- and minus-end directions. These findings illustrate the importance of a detailed mechanochemical model for dissecting the contributions of microscopic parameters to monomeric kinesin dynamics.

  View details for DOI 10.1088/0953-8984/23/37/374106

  View details for Web of Science ID 000294785400007

  View details for PubMedID 21862841

• The bacterial actin MreB rotates, and rotation depends on cell-wall assembly PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA van Teeffelen, S., Wang, S., Furchtgott, L., Huang, K. C., Wingreen, N. S., Shaevitz, J. W., Gitai, Z. 2011; 108 (38): 15822-15827

  Abstract

  Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.

  View details for DOI 10.1073/pnas.1108999108

  View details for Web of Science ID 000295030000036

  View details for PubMedID 21903929

  View details for PubMedCentralID PMC3179079

• Does the Potential for Chaos Constrain the Embryonic Cell-Cycle Oscillator? PLOS COMPUTATIONAL BIOLOGY McIsaac, R. S., Huang, K. C., Sengupta, A., Wingreen, N. S. 2011; 7 (7)

  Abstract

  Although many of the core components of the embryonic cell-cycle network have been elucidated, the question of how embryos achieve robust, synchronous cellular divisions post-fertilization remains unexplored. What are the different schemes that could be implemented by the embryo to achieve synchronization? By extending a cell-cycle model previously developed for embryos of the frog Xenopus laevis to include the spatial dimensions of the embryo, we establish a novel role for the rapid, fertilization-initiated calcium wave that triggers cell-cycle oscillations. Specifically, in our simulations a fast calcium wave results in synchronized cell cycles, while a slow wave results in full-blown spatio-temporal chaos. We show that such chaos would ultimately lead to an unpredictable patchwork of cell divisions across the embryo. Given this potential for chaos, our results indicate a novel design principle whereby the fast calcium-wave trigger following embryo fertilization synchronizes cell divisions.

  View details for DOI 10.1371/journal.pcbi.1002109

  View details for Web of Science ID 000293333200014

  View details for PubMedID 21779158

• Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria MOLECULAR MICROBIOLOGY Furchtgott, L., Wingreen, N. S., Huang, K. C. 2011; 81 (2): 340-353

  Abstract

  For the rod-shaped Gram-negative bacterium Escherichia coli, changes in cell shape have critical consequences for motility, immune system evasion, proliferation and adhesion. For most bacteria, the peptidoglycan cell wall is both necessary and sufficient to determine cell shape. However, how the synthesis machinery assembles a peptidoglycan network with a robustly maintained micron-scale shape has remained elusive. To explore shape maintenance, we have quantified the robustness of cell shape in three Gram-negative bacteria in different genetic backgrounds and in the presence of an antibiotic that inhibits division. Building on previous modelling suggesting a prominent role for mechanical forces in shape regulation, we introduce a biophysical model for the growth dynamics of rod-shaped cells to investigate the roles of spatial regulation of peptidoglycan synthesis, glycan-strand biochemistry and mechanical stretching during insertion. Our studies reveal that rod-shape maintenance requires insertion to be insensitive to fluctuations in cell-wall density and stress, and even a simple helical pattern of insertion is sufficient for over sixfold elongation without significant loss in shape. In addition, we demonstrate that both the length and pre-stretching of newly inserted strands regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shape-preserving cell-wall growth.

  View details for DOI 10.1111/j.1365-2958.2011.07616.x

  View details for Web of Science ID 000292567200007

  View details for PubMedID 21501250

  View details for PubMedCentralID PMC3134142

• Mechanics of membrane bulging during cell-wall disruption in Gram-negative bacteria PHYSICAL REVIEW E Daly, K. E., Huang, K. C., Wingreen, N. S., Mukhopadhyay, R. 2011; 83 (4)

  Abstract

  The bacterial cell wall is a network of sugar strands crosslinked by peptides that serve as the primary structure for bearing osmotic stress. Despite its importance in cellular survival, the robustness of the cell wall to network defects has been relatively unexplored. Treatment of the gram-negative bacterium Escherichia coli with the antibiotic vancomycin, which disrupts the crosslinking of new material during growth, leads to the development of pronounced bulges and eventually of cell lysis. Here, we model the mechanics of the bulging of the cytoplasmic membrane through pores in the cell wall. We find that the membrane undergoes a transition between a nearly flat state and a spherical bulge at a critical pore radius of ~20 nm. This critical pore size is large compared to the typical distance between neighboring peptides and glycan strands, and hence pore size acts as a constraint on network integrity. We also discuss the general implications of our model to membrane deformations in eukaryotic blebbing and vesiculation in red blood cells.

  View details for DOI 10.1103/PhysRevE.83.041922

  View details for Web of Science ID 000290152800013

  View details for PubMedID 21599215

• Bilayer-Mediated Clustering and Functional Interaction of MscL Channels BIOPHYSICAL JOURNAL Grage, S. L., Keleshian, A. M., Turdzeladze, T., Battle, A. R., Tay, W. C., May, R. P., Holt, S. A., Contera, S. A., Haertlein, M., Moulin, M., Pal, P., Rohde, P. R., Forsyth, V. T., Watts, A., Huang, K. C., Ulrich, A. S., Martinac, B. 2011; 100 (5): 1252-1260

  Abstract

  Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

  View details for DOI 10.1016/j.bpj.2011.01.023

  View details for Web of Science ID 000288049400013

  View details for PubMedID 21354398

• Mechanisms for Maintaining Cell-Shape in Rod-Shaped Gram-Negative Bacteria 55th Annual Meeting of the Biophysical-Society Furchtgott, L., Wingreen, N. S., Huang, K. C. CELL PRESS. 2011: 514–14

  View details for Web of Science ID 000306288604313

• Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, Y. E., Tropini, C., Jonas, K., Tsokos, C. G., Huang, K. C., Laub, M. T. 2011; 108 (3): 1052-1057

  Abstract

  Spatial asymmetry is crucial to development. One mechanism for generating asymmetry involves the localized synthesis of a key regulatory protein that diffuses away from its source, forming a spatial gradient. Although gradients are prevalent in eukaryotes, at both the tissue and intracellular levels, it is unclear whether gradients of freely diffusible proteins can form within bacterial cells given their small size and the speed of diffusion. Here, we show that the bacterium Caulobacter crescentus generates a gradient of the active, phosphorylated form of the master regulator CtrA, which directly regulates DNA replication. Using a combination of mathematical modeling, single-cell microscopy, and genetic manipulation, we demonstrate that this gradient is produced by the polarly localized phosphorylation and dephosphorylation of CtrA. Our data indicate that cells robustly establish the asymmetric fates of daughter cells before cell division causes physical compartmentalization. More generally, our results demonstrate that uniform protein abundance may belie gradients and other sophisticated spatial patterns of protein activity in bacterial cells.

  View details for DOI 10.1073/pnas.1015397108

  View details for Web of Science ID 000286310300033

  View details for PubMedID 21191097

• Entropy-driven translocation of an unstructured protein through the Gram-positive cell wall. Annual Meeting of the American-Society-for-Cell-Biology (ASCB) Halladin, D. K., Huang, K. C., Gopinathan, A., Theriot, J. A. AMER SOC CELL BIOLOGY. 2011

  View details for Web of Science ID 000305505503548

• Resolution limits of optical microscopy and the mind Biomed Comp Rev Usrell, T. S., Huang, K. C. 2011; 7: 27
• Clustering and functional interaction of MscL channels Biophys. J. Grage, L., Keleshian, A. M., Turdzeladze, T., Battle, A. R., Tay, W. C., May, R. P., Huang, K. 2011; 100: 1252-1260
• A spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium Proc Nat Acadsci USA. Selected for Feb 1, 2011 issue of Virtual Journal of Biological Physics Research. Chen, Y. E., Tropini, C., Huang, K. C., Laub, M. T. 2011; 108: 1052-1057
• The mechanics of membrane bulging during cell-wall disruption in Gram-negative bacteria Phys. Rev. Selected for May 1, 2011 issue of Virtual Journal of Biological Physics Research. Daly, K. E., Huang, K. C., Wingreen, N. S., Mukhopadhyay, R. 2011; 83: 041922
• Dynamic SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation GENES & DEVELOPMENT Fleming, T. C., Shin, J. Y., Lee, S., Becker, E., Huang, K. C., Bustamante, C., Pogliano, K. 2010; 24 (11): 1160-1172

  Abstract

  SpoIIIE is an FtsK-related protein that transports the forespore chromosome across the Bacillus subtilis sporulation septum. We use membrane photobleaching and protoplast assays to demonstrate that SpoIIIE is required for septal membrane fission in the presence of trapped DNA, and that DNA is transported across separate daughter cell membranes, suggesting that SpoIIIE forms a channel that partitions the daughter cell membranes. Our results reveal a close correlation between septal membrane fission and the assembly of a stable SpoIIIE translocation complex at the septal midpoint. Time-lapse epifluorescence, total internal reflection fluorescence (TIRF) microscopy, and live-cell photoactivation localization microscopy (PALM) demonstrate that the SpoIIIE transmembrane domain mediates dynamic localization to active division sites, whereas the assembly of a stable focus also requires the cytoplasmic domain. The transmembrane domain fails to completely separate the membrane, and it assembles unstable foci. TIRF microscopy and biophysical modeling of fluorescence recovery after photobleaching (FRAP) data suggest that this unstable protein transitions between disassembled and assembled oligomeric states. We propose a new model for the role of SpoIIIE assembly in septal membrane fission that has strong implications for how the chromosome terminus crosses the septum.

  View details for DOI 10.1101/gad.1925210

  View details for Web of Science ID 000278267200010

  View details for PubMedID 20516200

  View details for PubMedCentralID PMC2878653

• Macromolecules that prefer their membranes curvy MOLECULAR MICROBIOLOGY Huang, K. C., Ramamurthi, K. S. 2010; 76 (4): 822-832

  Abstract

  Understanding the mechanisms that underlie the organization of bacterial cells has become a significant challenge in the field of bacterial cytology. Of specific interest are early macromolecular sorting events that establish cellular non-uniformity and provide chemical landmarks for later localization events. In this review, we will examine specific examples of lipids and proteins that appear to exploit differences in membrane curvature to drive their localization to particular regions of a bacterial cell. We will also discuss the physical limits of curvature-mediated localization within bacteria, and the use of modelling to infer biophysical properties of curvature-sensing macromolecules.

  View details for DOI 10.1111/j.1365-2958.2010.07168.x

  View details for Web of Science ID 000277607600004

  View details for PubMedID 20444099

  View details for PubMedCentralID PMC2909655

• SpoIIIE assembly mediates septal membrane fission during Bacillus subtilis sporulation Genes and Development  Fleming, T., Becker, E., Lee, S., Shin, J. Y., Huang, K. C., Bustamante, C. 2010; 24: 1160
• Cell shape and cell-wall organization in Gram-negative bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huang, K. C., Mukhopadhyay, R., Wen, B., Gitai, Z., Wingreen, N. S. 2008; 105 (49): 19282-19287

  Abstract

  In bacterial cells, the peptidoglycan cell wall is the stress-bearing structure that dictates cell shape. Although many molecular details of the composition and assembly of cell-wall components are known, how the network of peptidoglycan subunits is organized to give the cell shape during normal growth and how it is reorganized in response to damage or environmental forces have been relatively unexplored. In this work, we introduce a quantitative physical model of the bacterial cell wall that predicts the mechanical response of cell shape to peptidoglycan damage and perturbation in the rod-shaped Gram-negative bacterium Escherichia coli. To test these predictions, we use time-lapse imaging experiments to show that damage often manifests as a bulge on the sidewall, coupled to large-scale bending of the cylindrical cell wall around the bulge. Our physical model also suggests a surprising robustness of cell shape to peptidoglycan defects, helping explain the observed porosity of the cell wall and the ability of cells to grow and maintain their shape even under conditions that limit peptide crosslinking. Finally, we show that many common bacterial cell shapes can be realized within the same model via simple spatial patterning of peptidoglycan defects, suggesting that minor patterning changes could underlie the great diversity of shapes observed in the bacterial kingdom.

  View details for DOI 10.1073/pnas.0805309105

  View details for Web of Science ID 000261706600048

  View details for PubMedID 19050072

• Lipid localization in bacterial cells through curvature-mediated microphase separation BIOPHYSICAL JOURNAL Mukhopadhyay, R., Huang, K. C., Wingreen, N. S. 2008; 95 (3): 1034-1049

  Abstract

  Although many proteins are known to localize in bacterial cells, for the most part our understanding of how such localization takes place is limited. Recent evidence that the phospholipid cardiolipin localizes to the poles of rod-shaped bacteria suggests that targeting of some proteins may rely on the heterogeneous distribution of membrane lipids. Membrane curvature has been proposed as a factor in the polar localization of high-intrinsic-curvature lipids, but the small size of lipids compared to the dimensions of the cell means that single molecules cannot stably localize. At the other extreme, phase separation of the membrane energetically favors a single domain of such lipids at one pole. We have proposed a physical mechanism in which osmotic pinning of the membrane to the cell wall naturally produces microphase separation, i.e., lipid domains of finite size, whose aggregate sensitivity to cell curvature can support spontaneous and stable localization to both poles. Here, we demonstrate that variations in the strength of pinning of the membrane to the cell wall can also act as a strong localization mechanism, in agreement with observations of cardiolipin relocalization from the poles to the septum during sporulation in the bacterium Bacillus subtilis. In addition, we rigorously determine the relationship between localization and the domain-size distribution including the effects of entropy, and quantify the strength of domain-domain interactions. Our model predicts a critical concentration of cardiolipin below which domains will not form and hence polar localization will not take place. This observation is consistent with recent experiments showing that in Escherichia coli cells with reduced cardiolipin concentrations, cardiolipin and the osmoregulatory protein ProP fail to localize to the poles.

  View details for DOI 10.1529/biophysj.107.126920

  View details for Web of Science ID 000257719200005

  View details for PubMedID 18390605

• The Min system as a general cell-geometry detection mechanism: patterns of Min oscillations respond to changes in cell shape in aberrantly shaped Escherichia coli J. Bacteriol Varma, A., Huang, K. C., Young, K. D. 2008; 190: 2106
• Cooperative gating and spatial organization of membrane proteins through elastic interactions PLOS COMPUTATIONAL BIOLOGY Ursell, T., Huang, K. C., Peterson, E., Phillips, R. 2007; 3 (5): 803-812

  Abstract

  Biological membranes are elastic media in which the presence of a transmembrane protein leads to local bilayer deformation. The energetics of deformation allow two membrane proteins in close proximity to influence each other's equilibrium conformation via their local deformations, and spatially organize the proteins based on their geometry. We use the mechanosensitive channel of large conductance (MscL) as a case study to examine the implications of bilayer-mediated elastic interactions on protein conformational statistics and clustering. The deformations around MscL cost energy on the order of 10 kBT and extend approximately 3 nm from the protein edge, as such elastic forces induce cooperative gating, and we propose experiments to measure these effects. Additionally, since elastic interactions are coupled to protein conformation, we find that conformational changes can severely alter the average separation between two proteins. This has important implications for how conformational changes organize membrane proteins into functional groups within membranes.

  View details for DOI 10.1371/journal.pcbi.0030081

  View details for Web of Science ID 000249105100004

  View details for PubMedID 17480116

• Control of melting using nanoscale coatings Huang, K. C., Wang, T., Joannopoulos, J. D. 2007
• Cooperative gating and spatial organization of membrane proteins through elastic interactions PLoS Comp. Biol. Ursell, T., Huang, K. C., Peterson, E., Phillips, R. 2007; 3: e81
• A curvature-mediated mechanism for localization of lipids to bacterial poles PLOS COMPUTATIONAL BIOLOGY Huang, K. C., Mukhopadhyay, R., Wingreen, N. S. 2006; 2 (11): 1357-1364

  Abstract

  Subcellular protein localization is a universal feature of eukaryotic cells, and the ubiquity of protein localization in prokaryotic species is now acquiring greater appreciation. Though some targeting anchors are known, the origin of polar and division-site localization remains mysterious for a large fraction of bacterial proteins. Ultimately, the molecular components responsible for such symmetry breaking must employ a high degree of self-organization. Here we propose a novel physical mechanism, based on the two-dimensional curvature of the membrane, for spontaneous lipid targeting to the poles and division site of rod-shaped bacterial cells. If one of the membrane components has a large intrinsic curvature, the geometrical constraint of the plasma membrane by the more rigid bacterial cell wall naturally leads to lipid microphase separation. We find that the resulting clusters of high-curvature lipids are large enough to spontaneously and stably localize to the two cell poles. Recent evidence of localization of the phospholipid cardiolipin to the poles of bacterial cells suggests that polar targeting of some proteins may rely on the membrane's differential lipid content. More generally, aggregates of lipids, proteins, or lipid-protein complexes may localize in response to features of cell geometry incapable of localizing individual molecules.

  View details for DOI 10.1371/journal.pcbi.0020151

  View details for Web of Science ID 000242375200005

  View details for PubMedID 17096591

• Nanoscale properties of melting at the surface of semiconductors PHYSICAL REVIEW B Huang, K. C., Wang, T., Joannopoulos, J. D. 2005; 72 (19)

  View details for DOI 10.1103/PhysRevB.72.195314

  View details for Web of Science ID 000233603700081

• Photonic band gaps and localization in the Thue-Morse structures APPLIED PHYSICS LETTERS Jiang, X. Y., Zhang, Y. G., Feng, S. L., Huang, K. C., Yi, Y. H., Joannopoulos, J. D. 2005; 86 (20)

  View details for DOI 10.1063/1.1928317

  View details for Web of Science ID 000229398000010

• Superheating and induced melting at semiconductor interfaces PHYSICAL REVIEW LETTERS Huang, K. C., Wang, T., Joannopoulos, J. D. 2005; 94 (17)

  Abstract

  We present ab initio density-functional simulations of the state of several semiconductor surfaces at temperatures near the bulk melting temperatures. We find that the solid-liquid phase-transition temperature at the surface can be altered via a microscopic (single-monolayer) coating with a different lattice-matched semiconducting material. Our results show that a single-monolayer GaAs coating on a Ge(110) surface above the Ge melting temperature can dramatically reduce the diffusion coefficient of the germanium atoms, going so far as to prevent melting of the bulk layers on the 10 ps time scale. In contrast, a single-monolayer coating of Ge on a GaAs(110) surface introduces defects into the bulk and induces melting of the top layer of GaAs atoms 300 K below the GaAs melting point. To our knowledge, these calculations represent the first ab initio investigation of the superheating and induced melting phenomena.

  View details for DOI 10.1103/PhysRevLett.94.175702

  View details for Web of Science ID 000228932200044

  View details for PubMedID 15904312

• Photonic Band-Gaps and Localization in the Thue-Morse Structures Appl. Phys. Lett. Selected for May 23, 2005 issue of Virtual Journal of Nanoscale Science & Technology. Jiang, X., Zhang, Y., Feng, S., Huang, K. C., Yi, Y., Joannopoulos, J. D. 2005; 86: 201110
• Min-protein oscillations in round bacteria PHYSICAL BIOLOGY Huang, K. C., Swingreen, N. S. 2004; 1 (4): 229-235

  Abstract

  In rod-shaped Escherichia coli cells, the Min proteins, which are involved in division-site selection, oscillate from pole-to-pole. The homologs of the Min proteins from the round bacterium Neisseria gonorrhoeae also form a spatial oscillator when expressed in wild-type and round, rodA- mutants of E. coli, suggesting that the Min proteins form an oscillator in N. gonorrhoeae. Here we report that a numerical model for Min-protein oscillations in rod-shaped cells also produces oscillations in round cells (cocci). Our numerical results explain why the MinE-protein rings found in wild-type E. coli are absent in round mutants. In addition, we find that for round cells there is a minimum radius below which oscillations do not occur, and a maximum radius above which oscillations become mislocalized. Finally, we demonstrate that Min-protein oscillations can select the long axis in nearly round cells based solely on geometry, a potentially important factor in division-plane selection in cocci.

  View details for DOI 10.1088/1478-3967/1/4/005

  View details for Web of Science ID 000234227100008

• Pattern formation within Escherichia coli: Diffusion, membrane attachment, and self-interaction of MinD molecules PHYSICAL REVIEW LETTERS Kulkarni, R. V., Huang, K. C., Kloster, M., Wingreen, N. S. 2004; 93 (22)

  Abstract

  In E. coli, accurate cell division depends upon the oscillation of Min proteins from pole to pole. We provide a model for the polar localization of MinD based only on diffusion, a delay for nucleotide exchange, and different rates of attachment to the bare membrane and the occupied membrane. We derive analytically the probability density, and correspondingly the length scale, for MinD attachment zones. Our simple analytical model illustrates the processes giving rise to the observed localization of cellular MinD zones.

  View details for DOI 10.1103/PhysRevLett.93.228103

  View details for Web of Science ID 000225326300053

  View details for PubMedID 15601121

• Negative effective permeability in polaritonic photonic crystals APPLIED PHYSICS LETTERS Huang, K. C., Povinelli, M. L., Joannopoulos, J. D. 2004; 85 (4): 543-545

  View details for DOI 10.1063/1.1775291

  View details for Web of Science ID 000222855400011

• Nature of lossy Bloch states in polaritonic photonic crystals PHYSICAL REVIEW B Huang, K. C., Lidorikis, E., Jiang, X. Y., Joannopoulos, J. D., Nelson, K. A., Bienstman, P., Fan, S. H. 2004; 69 (19)

  View details for DOI 10.1103/PhysRevB.69.195111

  View details for Web of Science ID 000221961700034

• Pattern Formation within Escherichia coli: Diffusion, Membrane Attachment, and Self-Interaction of MinD Molecules Phys. Rev. Lett. Selected for December 1, 2004 issue of Virtual Journal of Biological Physics. Kulkarni, R. V., Huang, K. C., Kloster, M., Wingreen, N. S. 2004; 93: 228103
• The nature of lossy Bloch states in polaritonic photonic crystals Phys. Rev. Selected for June 7, 2004 issue of Virtual Journal of Nanoscale Science & Technology. Huang, K. C., Lidorikis, E., Jiang, X., Joannopoulos, J. D., Nelson, K. A., Bienstman, P. 2004; B 69: 195111
• Dynamic structures in Escherichia coli: Spontaneous formation of MinE rings and MinD polar zones PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huang, K. C., Meir, Y., Wingreen, N. S. 2003; 100 (22): 12724-12728

  Abstract

  In Escherichia coli, division site selection is regulated in part by the Min-protein system. Oscillations of the Min proteins from pole to pole every approximately 40 sec have been revealed by in vivo studies of GFP fusions. The dynamic oscillatory structures produced by the Min proteins, including a ring of MinE protein, compact polar zones of MinD, and zebra-striped oscillations in filamentous cells, remain unexplained. We show that the Min oscillations, including mutant phenotypes, can be accounted for by in vitro-observed interactions involving MinD and MinE, with a crucial role played by the rate of nucleotide exchange. Recent discoveries suggest that protein oscillations may play a general role in proper chromosome and plasmid partitioning.

  View details for DOI 10.1073/pnas.2135445100

  View details for Web of Science ID 000186301100040

  View details for PubMedID 14569005

• Phonon-polariton excitations in photonic crystals PHYSICAL REVIEW B Huang, K. C., Bienstman, P., Joannopoulos, J. D., Nelson, K. A., Fan, S. 2003; 68 (7)

  View details for DOI 10.1103/PhysRevB.68.075209

  View details for Web of Science ID 000185241200057

• Field expulsion and reconfiguration in polaritonic photonic crystals PHYSICAL REVIEW LETTERS Huang, K. C., Bienstman, P., Joannopoulos, J. D., Nelson, K. A., Fan, S. H. 2003; 90 (19)

  Abstract

  We uncover a rich set of optical phenomena stemming from the incorporation of polar materials exhibiting transverse phonon polariton excitations into a photonic crystal structure. We identify in the frequency spectrum two regimes in which the dielectric response of the polaritonic medium can induce extreme localization of the electromagnetic energy. Our analysis of the effect of polarization and the interaction between the polariton and photonic band gaps on the Bloch states leads to a pair of mechanisms for sensitive frequency-controlled relocation and/or reconfiguration of the fields.

  View details for DOI 10.1103/PhysRevLett.90.196402

  View details for Web of Science ID 000182928300030

  View details for PubMedID 12785962

• Isolation and preparation of bacterial cell walls for Ultra-Performance Liquid Chromatography in press, J Vis Exp. Desmarais, S., Cava, F., de Pedro, M., Huang, K. C.
• Comment on "Quantum Monte Carlo study of the dipole moment of CO" J. Chem. Phys. Huang, K. C., Needs, R. J., Rajagopal, G. 1999, 2000; 110, 112: 11700, 4419