Doctor of Philosophy, Stanford University, BIOPH-PHD (2014)
Bachelor of Science, University of British Columbia, Biophysics (2008)
The Gut Microbiome: Connecting Spatial Organization to Function
CELL HOST & MICROBE
2017; 21 (4): 433-442
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
- High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography JOURNAL OF BIOLOGICAL CHEMISTRY 2015; 290 (52): 31090-31100
- Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations CELL REPORTS 2014; 9 (4): 1520-1527
Principles of bacterial cell-size determination revealed by cell-wall synthesis perturbations.
2014; 9 (4): 1520-1527
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
A dynamically assembled cell wall synthesis machinery buffers cell growth.
Proceedings of the National Academy of Sciences of the United States of America
2014; 111 (12): 4554-4559
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
Physical constraints on the establishment of intracellular spatial gradients in bacteria
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
Interplay between the Localization and Kinetics of Phosphorylation in Flagellar Pole Development of the Bacterium Caulobacter crescentus
PLOS COMPUTATIONAL BIOLOGY
2012; 8 (8)
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
Measuring the stiffness of bacterial cells from growth rates in hydrogels of tunable elasticity
2012; 84 (5): 874-891
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
Islands Containing Slowly Hydrolyzable GTP Analogs Promote Microtubule Rescues
2012; 7 (1)
Microtubules are dynamic polymers of GTP- and GDP-tubulin that undergo stochastic transitions between growing and shrinking phases. Rescues, the conversion from shrinking to growing, have recently been proposed to be to the result of regrowth at GTP-tubulin islands within the lattice of growing microtubules. By introducing mixed GTP/GDP/GMPCPP (GXP) regions within the lattice of dynamic microtubules, we reconstituted GXP islands in vitro (GMPCPP is the slowly hydrolyzable GTP analog guanosine-5'-[(α,β)-methyleno]triphosphate). We found that such islands could reproducibly induce rescues and that the probability of rescue correlated with both the size of the island and the percentage of GMPCPP-tubulin within the island. The islands slowed the depolymerization rate of shortening microtubules and promoted regrowth more readily than GMPCPP seeds. Together, these findings provide new mechanistic insights supporting the possibility that rescues could be triggered by enriched GTP-tubulin regions and present a new tool for studying such rescue events in vitro.
View details for DOI 10.1371/journal.pone.0030103
View details for Web of Science ID 000301454400059
View details for PubMedID 22272281
Megapixel digital PCR
2011; 8 (8): 649-U64
We present a microfluidic 'megapixel' digital PCR device that uses surface tension-based sample partitioning and dehydration control to enable high-fidelity single DNA molecule amplification in 1,000,000 reactors of picoliter volume with densities up to 440,000 reactors cm(-2). This device achieves a dynamic range of 10(7), single-nucleotide-variant detection below one copy per 100,000 wild-type sequences and the discrimination of a 1% difference in chromosome copy number.
View details for DOI 10.1038/NMETH.1640
View details for Web of Science ID 000293220600018
View details for PubMedID 21725299
Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (3): 1052-1057
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
Nonexponential Kinetics of DNA Escape from alpha-Hemolysin Nanopores
2008; 95 (11): 5317-5323
Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.
View details for DOI 10.1529/biophysj.108.137760
View details for Web of Science ID 000260999500031
View details for PubMedID 18775965
Multi-nanopore force Spectroscopy for DNA analysis
2007; 92 (5): 1632-1637
The need for low-cost DNA sequence detection in clinical applications is driving development of new technologies. We demonstrate a method for detection of mutations in a DNA sequence purely by electronic means, and without need for fluorescent labeling. Our method uses an array of nanopores to perform synchronized single-molecule force spectroscopy measurements over many molecules in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement.
View details for DOI 10.1529/biophysj.106.094060
View details for Web of Science ID 000244373800018
View details for PubMedID 17158571