Youngbin Lim

All Publications

• Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature communications Lim, Y., Bak, S. Y., Sung, K., Jeong, E., Lee, S. H., Kim, J. S., Bae, S., Kim, S. K. 2016; 7: 13350

  Abstract

  The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.

  View details for DOI 10.1038/ncomms13350

  View details for PubMedID 27804953

• Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model. Cell stem cell Indana, D., Zakharov, A., Lim, Y., Dunn, A. R., Bhutani, N., Shenoy, V. B., Chaudhuri, O. 2024; 31 (5): 640-656.e8

  Abstract

  Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 μm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

  View details for DOI 10.1016/j.stem.2024.03.016

  View details for PubMedID 38701758

• Mechanical expansion microscopy. Methods in cell biology Fan, Y. n., Lim, Y. n., Wyss, L. S., Park, S. n., Xu, C. n., Fu, H. n., Fei, J. n., Hong, Y. n., Wang, B. n. 2021; 161: 125–46

  Abstract

  This chapter describes two mechanical expansion microscopy methods with accompanying step-by-step protocols. The first method, mechanically resolved expansion microscopy, uses non-uniform expansion of partially digested samples to provide the imaging contrast that resolves local mechanical properties. Examining bacterial cell wall with this method, we are able to distinguish bacterial species in mixed populations based on their distinct cell wall rigidity and detect cell wall damage caused by various physiological and chemical perturbations. The second method is mechanically locked expansion microscopy, in which we use a mechanically stable gel network to prevent the original polyacrylate network from shrinking in ionic buffers. This method allows us to use anti-photobleaching buffers in expansion microscopy, enabling detection of novel ultra-structures under the optical diffraction limit through super-resolution single molecule localization microscopy on bacterial cells and whole-mount immunofluorescence imaging in thick animal tissues. We also discuss potential applications and assess future directions.

  View details for DOI 10.1016/bs.mcb.2020.04.013

  View details for PubMedID 33478686

• 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

• Recent advancement of light-based single-molecule approaches for studying biomolecules WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE Croop, B., Zhang, C., Lim, Y., Gelfand, R. M., Han, K. 2019; 11 (4)

  View details for DOI 10.1002/wsbm.1445

  View details for Web of Science ID 000471695000001

• Incorporation of STED technique into single-molecule spectroscopy to break the concentration limit of diffusing molecules in single-molecule detection CHEMICAL COMMUNICATIONS Kim, N., Kwon, J., Lim, Y., Kang, J., Bae, S., Kim, S. 2018; 54 (69): 9667–70

  Abstract

  By incorporating STED (stimulated emission depletion) nanoscopy into single-molecule spectroscopy, we demonstrate that the concentration limit imposed by optical diffraction can be overcome in diffusion-based single-molecule measurement. We showed that single-molecule detection is feasible at a concentration of 5 nM, which is 100-times higher than the limit of conventional single-molecule measurements.

  View details for DOI 10.1039/c8cc05726e

  View details for Web of Science ID 000442605100026

  View details for PubMedID 30101240

• Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, M., Kim, H. J., Son, S. H., Yoon, H. J., Lim, Y., Lee, J. W., Seok, Y., Jin, K. S., Yu, Y. G., Kim, S. K., Ryu, S., Lee, H. H. 2016; 113 (18): E2480-E2488

  Abstract

  DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92-198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant.

  View details for DOI 10.1073/pnas.1602618113

  View details for Web of Science ID 000375395700005

  View details for PubMedID 27099293

• New sub-diffraction-limit microscopy technique: Dual-point illumination AND-gate microscopy on nanodiamonds (DIAMOND) OPTICS EXPRESS Kwon, J., Lim, Y., Jung, J., Kim, S. K. 2012; 20 (12): 13347-13356

  Abstract

  We introduce a new, easily implementable sub-diffraction-limit microscopy technique utilizing the optical AND-gate property of fluorescent nanodiamond (FND). We demonstrate that when FND is illuminated by two spatially-offset lights of different wavelengths, emission comes only from the region of their overlap, which is used to reduce the effective point spread function from ~300 nm to ~130 nm in lateral plane, well below the diffraction limit.

  View details for DOI 10.1364/OE.20.013347

  View details for Web of Science ID 000305463600072

  View details for PubMedID 22714363