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KC Huang
Professor of Bioengineering and of Microbiology and Immunology
My laboratory employs diverse interdisciplinary methods of inquiry to understand the relationships among cell shape detection, determination, and maintenance in bacteria. Cell shape plays a critical role in regulating many physiological functions, yet little is known about how the wide variety of cell shapes are determined and maintained. Inside the cell, many proteins organize and segregate, but how they detect and respond to the cellular morphology to end up at the right place at the right time is also largely mysterious. The group uses a combination of analytical, computational, and experimental approaches to probe physical mechanisms of shape-related self-organization in protein networks, membranes, and the cell wall. Current topics of interest are (i) cell-wall biosynthesis, (ii) the regulation and mechanics of cell division, (iii) membrane organization, and (iv) membrane-mediated protein interactions. Ultimately, the manipulation of cell shape may provide a direct tool for engineering complex cellular behaviors.
Professor, Bioengineering
Professor, Microbiology & Immunology
Member, Bio-X
Faculty Fellow, Stanford ChEM-H
CAREER Award, National Science Foundation (2012-2017)
NIH Director's New Innovator Award, National Institutes of Health (2009-2014)
Helen Hay Whitney Fellowship, Helen Hay Whitney Foundation (2005-2008)
Ph. D., MIT, Physics (2004)
M. Phil., Cambridge University, Physics (1999)
B.S., Caltech, Physics/Mathematics (1998)
Academic kchuang@stanford.edu
Alternate Contact Jennifer Gucwa Administrative Assistant jennifer.gucwa@stanford.edu
Curriculum Vitae PDF
NIH Biosketch PDF
Laboratory of Cellular Organization
Current Research and Scholarly Interests
We primarily focus on bacteria, in which the exquisite patterning of the interior in both space and time is critical for a wide variety of cellular functions. The wide variety of shapes and sizes that bacteria take on can be used as synthetic environment for studying the establishment of intracellular organization and the cellular response to perturbations in morphology. Ultimately, the manipulation of cell shape may provide a direct tool for engineering complex cellular behaviors.
Currently, we are interested in (i) the role of the cell wall in cell-shape determination, (ii) the regulation and mechanics of the cell cycle and cell division, (iii) the spatial and temporal organization of the membrane, (iv) the role of the membrane in transmembrane-protein interactions and ion channel gating, and (v) collective behavior in bacteria.
2019-20 Courses
Physical Biology
BIOE 42 (Spr)
Independent Studies (11)
Bioengineering Problems and Experimental Investigation
BIOE 191 (Aut, Win, Spr, Sum)
Directed Investigation
Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Sum)
Directed Reading in Microbiology and Immunology
MI 299 (Win, Spr)
BIOPHYS 300 (Aut, Win, Spr, Sum)
MI 399 (Aut, Win, Spr, Sum)
Medical Scholars Research
BIOE 299B (Sum)
Writing of Original Research for Engineers
ENGR 199W (Spr)
Prior Year Courses
Advanced Seminar on Prokaryotic Molecular Biology
BIO 346 (Aut)
Gut Microbiota in Health and Disease
BIOE 221G, MI 221 (Spr)
Physical Biology of Cells
Stanford Advisees
Doctoral Dissertation Reader (AC)
Katie Bodner, Heejo Choi, Bryan Merrill, Kali Pruss, Tim Schnabel, Will Van Treuren
Postdoctoral Faculty Sponsor
Heidi Arjes, Po-Yi Ho, Manohary Rajendram, Anthony Shiver
Doctoral Dissertation Advisor (AC)
Andres Aranda-Diaz, Esha Atolia, Spencer Cesar, Rebecca Culver, Ben Knapp, Kim Vasquez
Postdoctoral Research Mentor
Heidi Arjes, Manohary Rajendram
Doctoral (Program)
Rafi Ayub, Phillip DiGiacomo, Thomas Lozanoski, Caitlin Maikawa, Taylor Nguyen, Sam Vesuna, Linfeng Yang
Graduate and Fellowship Programs
Biochemistry (Phd Program)
Biophysics (Phd Program)
Microbiology and Immunology (Phd Program)
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.
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
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
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
Emergent Phototactic Responses of Cyanobacteria under Complex Light Regimes MBIO Chau, R. M., Bhaya, D., Huang, K. C. 2017; 8 (2)
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
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-?
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 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
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
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
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.
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
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
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
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
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
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
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)
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.
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
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
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
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.
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
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
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
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
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
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
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 REG3?. 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.
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
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
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 (?CTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with ?-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 ?CTL; however, cells expressing ?CTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ?CTL 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
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
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
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
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
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
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
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 ?-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.
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
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.
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)
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
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)
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
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
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
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
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.
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
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.
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
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
How and why cells grow as rods BMC BIOLOGY Chang, F., Huang, K. C. 2014; 12
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
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.
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
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.
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
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.
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
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
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.
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
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
The role of hydrolases in bacterial cell-wall growth. Current opinion in microbiology Lee, T. K., Huang, K. C. 2013; 16 (6): 760-766
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.
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
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
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)
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
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
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
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.
Optimal Dynamics for Quality Control in Spatially Distributed Mitochondrial Networks PLOS COMPUTATIONAL BIOLOGY Patel, P. K., Shirihai, O., Huang, K. C. 2013; 9 (7)
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.
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
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
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.
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.
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
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.
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
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.
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)
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.
Physical constraints on the establishment of intracellular spatial gradients in bacteria BMC BIOPHYSICS Tropini, C., Rabbani, N., Huang, K. C. 2012; 5
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
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)
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.
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
Microtubules are built from linear polymers of ?-? 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 ?-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 ?-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 ?-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
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
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.
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
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
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
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.
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)
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
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
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.
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)
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.
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
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.
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)
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
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
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.
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
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
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.
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
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
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
Macromolecules that prefer their membranes curvy MOLECULAR MICROBIOLOGY Huang, K. C., Ramamurthi, K. S. 2010; 76 (4): 822-832
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.
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
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.
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
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
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
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.
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
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.
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
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
Superheating and induced melting at semiconductor interfaces PHYSICAL REVIEW LETTERS Huang, K. C., Wang, T., Joannopoulos, J. D. 2005; 94 (17)
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
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
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
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)
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.
Negative effective permeability in polaritonic photonic crystals APPLIED PHYSICS LETTERS Huang, K. C., Povinelli, M. L., Joannopoulos, J. D. 2004; 85 (4): 543-545
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)
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
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.
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)
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)
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.
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