Publications by authors named "Julie A Theriot"

89 Publications

Actin cables and comet tails organize mitochondrial networks in mitosis.

Nature 2021 Mar 3;591(7851):659-664. Epub 2021 Mar 3.

Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.
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http://dx.doi.org/10.1038/s41586-021-03309-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990722PMC
March 2021

Elastic wrinkling of keratocyte lamellipodia driven by myosin-induced contractile stress.

Biophys J 2021 Feb 23. Epub 2021 Feb 23.

Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, Washington. Electronic address:

During actin-based cell migration, the actin cytoskeleton in the lamellipodium both generates and responds to force, which has functional consequences for the ability of the cell to extend protrusions. However, the material properties of the lamellipodial actin network and its response to stress on the timescale of motility are incompletely understood. Here, we describe a dynamic wrinkling phenotype in the lamellipodium of fish keratocytes, in which the actin sheet buckles upward away from the ventral membrane of the cell, forming a periodic pattern of wrinkles perpendicular to the cell's leading edge. Cells maintain an approximately constant wrinkle wavelength over time despite new wrinkle formation and the lateral movement of wrinkles in the cell frame of reference, suggesting that cells have a preferred or characteristic wrinkle wavelength. Generation of wrinkles is dependent upon myosin contractility, and their wavelength scales directly with the density of the actin network and inversely with cell adhesion. These results are consistent with a simple physical model for wrinkling in an elastic sheet under compression and suggest that the lamellipodial cytoskeleton behaves as an elastic material on the timescale of cell migration despite rapid actin turnover.
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http://dx.doi.org/10.1016/j.bpj.2021.02.022DOI Listing
February 2021

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially infected epithelial cells.

Dev Cell 2021 Feb;56(4):443-460.e11

Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Electronic address:

Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.
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http://dx.doi.org/10.1016/j.devcel.2021.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982222PMC
February 2021

Osmolarity-independent electrical cues guide rapid response to injury in zebrafish epidermis.

Elife 2020 11 23;9. Epub 2020 Nov 23.

Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, United States.

The ability of epithelial tissues to heal after injury is essential for animal life, yet the mechanisms by which epithelial cells sense tissue damage are incompletely understood. In aquatic organisms such as zebrafish, osmotic shock following injury is believed to be an early and potent activator of a wound response. We find that, in addition to sensing osmolarity, basal skin cells in zebrafish larvae are also sensitive to changes in the particular ionic composition of their surroundings after wounding, specifically the concentration of sodium chloride in the immediate vicinity of the wound. This sodium chloride-specific wound detection mechanism is independent of cell swelling, and instead is suggestive of a mechanism by which cells sense changes in the transepithelial electrical potential generated by the transport of sodium and chloride ions across the skin. Consistent with this hypothesis, we show that electric fields directly applied within the skin are sufficient to initiate actin polarization and migration of basal cells in their native epithelial context in vivo, even overriding endogenous wound signaling. This suggests that, in order to mount a robust wound response, skin cells respond to both osmotic and electrical perturbations arising from tissue injury.
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http://dx.doi.org/10.7554/eLife.62386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721437PMC
November 2020

Cell Mechanics at the Rear Act to Steer the Direction of Cell Migration.

Cell Syst 2020 09 10;11(3):286-299.e4. Epub 2020 Sep 10.

Courant Institute of Mathematical Sciences and Department of Biology, New York University, New York, NY 10012, USA. Electronic address:

Motile cells navigate complex environments by changing their direction of travel, generating left-right asymmetries in their mechanical subsystems to physically turn. Currently, little is known about how external directional cues are propagated along the length scale of the whole cell and integrated with its force-generating apparatus to steer migration mechanically. We examine the mechanics of spontaneous cell turning in fish epidermal keratocytes and find that the mechanical asymmetries responsible for turning behavior predominate at the rear of the cell, where there is asymmetric centripetal actin flow. Using experimental perturbations, we identify two linked feedback loops connecting myosin II contractility, adhesion strength and actin network flow in turning cells that are sufficient to explain the observed cell shapes and trajectories. Notably, asymmetries in actin polymerization at the cell leading edge play only a minor role in the mechanics of cell turning-that is, cells steer from the rear.
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http://dx.doi.org/10.1016/j.cels.2020.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530145PMC
September 2020

A mechanical perspective on phagocytic cup formation.

Curr Opin Cell Biol 2020 10 19;66:112-122. Epub 2020 Jul 19.

Department of Biology, University of Washington, Seattle, WA 98105, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98105, USA. Electronic address:

Phagocytosis is a widespread and evolutionarily conserved process with diverse biological functions, ranging from engulfment of invading microbes during infection to clearance of apoptotic debris in tissue homeostasis. Along with differences in biochemical composition, phagocytic targets greatly differ in physical attributes, such as size, shape, and rigidity, which are now recognized as important regulators of this process. Force exertion at the cell-target interface and cellular mechanical changes during phagocytosis are emerging as crucial factors underlying sensing of such target properties. With technological developments, mechanical aspects of phagocytosis are increasingly accessible experimentally, revealing remarkable organizational complexity of force exertion. An increasingly high-resolution picture is emerging of how target physical cues and cellular mechanical properties jointly govern important steps throughout phagocytic engulfment.
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http://dx.doi.org/10.1016/j.ceb.2020.05.011DOI Listing
October 2020

Rapidly dynamic host cell heterogeneity in bacterial adhesion governs susceptibility to infection by .

Mol Biol Cell 2020 09 17;31(19):2097-2106. Epub 2020 Jun 17.

Department of Biology, University of Washington, Seattle, WA 98185-1800.

Interactions between host cells and individual pathogenic bacteria determine the clinical severity of disease during systemic infection in humans. Vascular endothelial cells, which line the lumen of blood vessels, represent a critical barrier for a bacterium in the bloodstream. These cells adopt a myriad of phenotypes that may modulate their susceptibility to infection; however, the precise determinants of their heterogeneity in susceptibility are not known. Here, we show that heterogeneity in susceptibility to infection among primary human vascular endothelial cells can be attributed entirely to robust, preexisting host cell heterogeneity in bacterial adhesion, and we find no evidence for significant heterogeneity in later steps of infection. High susceptibility to adhesion decays rapidly, within 30-60 min. Thus, rapidly fluctuating, nongenetic variability in bacterial adhesion diversifies susceptibility to infection, both among host cells and within individual cells over time.
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http://dx.doi.org/10.1091/mbc.E19-08-0454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530904PMC
September 2020

Neutrophil-like HL-60 cells expressing only GFP-tagged β-actin exhibit nearly normal motility.

Cytoskeleton (Hoboken) 2020 05 28;77(5-6):181-196. Epub 2020 Feb 28.

Department of Biology, Howard Hughes Medical Institute, University of Washington, Seattle, WA.

Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil-like HL-60 cell lines expressing GFP-β-actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL-60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal β-actin. Surprisingly, we recovered viable monoallelic GFP-β-actin cells as well as biallelic edited GFP-β-actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP-β-actin incorporates into the F-actin network in biallelic edited cells at similar efficiencies as normal β-actin in unedited cells. Additionally, the edited cells significantly upregulate γ-actin, perhaps helping to compensate for the loss of normal β-actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well-suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.
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http://dx.doi.org/10.1002/cm.21603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7383899PMC
May 2020

Microparticle traction force microscopy reveals subcellular force exertion patterns in immune cell-target interactions.

Nat Commun 2020 01 7;11(1):20. Epub 2020 Jan 7.

Department of Biochemistry, Stanford University, Stanford, CA, 94305, USA.

Force exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing spatial force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy for studying cellular interactions. We establish a straightforward batch approach for synthesizing uniform, deformable and tuneable hydrogel particles, which can also be easily derivatized. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a reference-free computational method allowing inference of traction forces with high sensitivity directly from the particle shape. We illustrate the potential of this approach by revealing subcellular force patterns throughout phagocytic engulfment and force dynamics in the cytotoxic T-cell immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications.
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http://dx.doi.org/10.1038/s41467-019-13804-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946705PMC
January 2020

Subendothelial stiffness alters endothelial cell traction force generation while exerting a minimal effect on the transcriptome.

Sci Rep 2019 12 3;9(1):18209. Epub 2019 Dec 3.

Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, WA, 98195-1800, USA.

Endothelial cells respond to changes in subendothelial stiffness by altering their migration and mechanics, but whether those responses are due to transcriptional reprogramming remains largely unknown. We measured traction force generation and also performed gene expression profiling for two endothelial cell types grown in monolayers on soft or stiff matrices: primary human umbilical vein endothelial cells (HUVEC) and immortalized human microvascular endothelial cells (HMEC-1). Both cell types respond to changes in subendothelial stiffness by increasing the traction stresses they exert on stiffer as compared to softer matrices, and exhibit a range of altered protein phosphorylation or protein conformational changes previously implicated in mechanotransduction. However, the transcriptome has only a minimal role in this conserved biomechanical response. Only few genes were differentially expressed in each cell type in a stiffness-dependent manner, and none were shared between them. In contrast, thousands of genes were differentially regulated in HUVEC as compared to HMEC-1. HUVEC (but not HMEC-1) upregulate expression of TGF-β2 on stiffer matrices, and also respond to application of exogenous TGF-β2 by enhancing their endogenous TGF-β2 expression and their cell-matrix traction stresses. Altogether, these findings provide insights into the relationship between subendothelial stiffness, endothelial mechanics and variation of the endothelial cell transcriptome, and reveal that subendothelial stiffness, while critically altering endothelial cells' mechanical behavior, minimally affects their transcriptome.
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http://dx.doi.org/10.1038/s41598-019-54336-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890669PMC
December 2019

Efficient Front-Rear Coupling in Neutrophil Chemotaxis by Dynamic Myosin II Localization.

Dev Cell 2019 04;49(2):189-205.e6

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Efficient chemotaxis requires rapid coordination between different parts of the cell in response to changing directional cues. Here, we investigate the mechanism of front-rear coordination in chemotactic neutrophils. We find that changes in the protrusion rate at the cell front are instantaneously coupled to changes in retraction at the cell rear, while myosin II accumulation at the rear exhibits a reproducible 9-15-s lag. In turning cells, myosin II exhibits dynamic side-to-side relocalization at the cell rear in response to turning of the leading edge and facilitates efficient turning by rapidly re-orienting the rear. These manifestations of front-rear coupling can be explained by a simple quantitative model incorporating reversible actin-myosin interactions with a rearward-flowing actin network. Finally, the system can be tuned by the degree of myosin regulatory light chain (MRLC) phosphorylation, which appears to be set in an optimal range to balance persistence of movement and turning ability.
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http://dx.doi.org/10.1016/j.devcel.2019.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708378PMC
April 2019

cell-to-cell spread in epithelia is heterogeneous and dominated by rare pioneer bacteria.

Elife 2019 02 5;8. Epub 2019 Feb 5.

Department of Biochemistry, Howard Hughes Medical Institute, Stanford University, Stanford, United States.

hijacks host actin to promote its intracellular motility and intercellular spread. While virulence hinges on cell-to-cell spread, little is known about the dynamics of bacterial spread in epithelia at a population level. Here, we use live microscopy and statistical modeling to demonstrate that cell-to-cell spread proceeds anisotropically in an epithelial monolayer in culture. We show that boundaries of infection foci are irregular and dominated by rare pioneer bacteria that spread farther than the rest. We extend our quantitative model for bacterial spread to show that heterogeneous spreading behavior can improve the chances of creating a persistent infection in an actively extruding epithelium. Thus, our results indicate that cell-to-cell spread is heterogeneous, and that rare pioneer bacteria determine the frontier of infection foci and may promote bacterial infection persistence in dynamic epithelia.

Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
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http://dx.doi.org/10.7554/eLife.40032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363384PMC
February 2019

Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum.

Nat Chem Biol 2019 03 21;15(3):221-231. Epub 2019 Jan 21.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.

Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.
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http://dx.doi.org/10.1038/s41589-018-0206-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693342PMC
March 2019

Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens.

Nat Genet 2018 12 5;50(12):1716-1727. Epub 2018 Nov 5.

Department of Genetics and Stanford University Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University School of Medicine, Stanford, CA, USA.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.
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http://dx.doi.org/10.1038/s41588-018-0254-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719718PMC
December 2018

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.

J Vis Exp 2018 07 5(137). Epub 2018 Jul 5.

Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.
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http://dx.doi.org/10.3791/57361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124605PMC
July 2018

The outer membrane is an essential load-bearing element in Gram-negative bacteria.

Nature 2018 07 18;559(7715):617-621. Epub 2018 Jul 18.

Department of Bioengineering, Stanford University, Stanford, CA, USA.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier that defines cell shape and allows the cell to sustain large mechanical loads such as turgor pressure. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.
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http://dx.doi.org/10.1038/s41586-018-0344-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6089221PMC
July 2018

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

PLoS Pathog 2018 05 30;14(5):e1007094. Epub 2018 May 30.

Benioff Children's Hospital, University of California, San Francisco, San Francisco, California, United States of America.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin's involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.
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http://dx.doi.org/10.1371/journal.ppat.1007094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044554PMC
May 2018

Surface Area to Volume Ratio: A Natural Variable for Bacterial Morphogenesis.

Trends Microbiol 2018 10 26;26(10):815-832. Epub 2018 May 26.

Department of Biochemistry, Department of Microbiology & Immunology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.

An immediately observable feature of bacteria is that cell size and shape are remarkably constant and characteristic for a given species in a particular condition, but vary quantitatively with physiological parameters such as growth rate, indicating both genetic and environmental regulation. However, despite decades of research, the molecular mechanisms underlying bacterial morphogenesis have remained incompletely characterized. We recently demonstrated that a wide range of bacterial species exhibit a robust surface area to volume ratio (SA/V) homeostasis. Because cell size, shape, and SA/V are mathematically interconnected, if SA/V is indeed the natural variable that cells actively monitor, this finding has critical implications for our understanding of bacterial morphogenesis, placing fundamental constraints on the sizes and shapes that cells can adopt. In this Opinion article we discuss the broad implications that this novel perspective has for the field of bacterial growth and morphogenesis.
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http://dx.doi.org/10.1016/j.tim.2018.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150810PMC
October 2018

Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin.

Mol Biol Cell 2018 07 2;29(13):1571-1589. Epub 2018 May 2.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305.

Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been studied previously, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens is hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm to host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.
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http://dx.doi.org/10.1091/mbc.E18-04-0228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080647PMC
July 2018

Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs.

Angew Chem Int Ed Engl 2018 05 14;57(19):5267-5272. Epub 2018 Apr 14.

Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.

Front-line tuberculosis (TB) drugs have been characterized extensively in vitro and in vivo with respect to gene expression and cell viability. However, little work has been devoted to understanding their effects on the physiology of the cell envelope, one of the main targets of this clinical regimen. Herein, we use metabolic labeling methods to visualize the effects of TB drugs on cell envelope dynamics in mycobacterial species. We developed a new fluorophore-trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super-resolution microscopy. We also probed the relationship between mycomembrane and peptidoglycan dynamics using a dual metabolic labeling strategy. Finally, we found that metabolic labeling of both cell envelope structures reports on drug effects on cell physiology in two hours, far faster than a genetic sensor of cell envelope stress. Our work provides insight into acute drug effects on cell envelope biogenesis in live mycobacteria.
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http://dx.doi.org/10.1002/anie.201712020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924460PMC
May 2018

Homeostatic Cell Growth Is Accomplished Mechanically through Membrane Tension Inhibition of Cell-Wall Synthesis.

Cell Syst 2017 12 1;5(6):578-590.e6. Epub 2017 Dec 1.

Department of Biochemistry and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Feedback mechanisms are required to coordinate balanced synthesis of subcellular components during cell growth. However, these coordination mechanisms are not apparent at steady state. Here, we elucidate the interdependence of cell growth, membrane tension, and cell-wall synthesis by observing their rapid re-coordination after osmotic shocks in Gram-positive bacteria. Single-cell experiments and mathematical modeling demonstrate that mechanical forces dually regulate cell growth: while turgor pressure produces mechanical stress within the cell wall that promotes its expansion through wall synthesis, membrane tension induces growth arrest by inhibiting wall synthesis. Tension inhibition occurs concurrently with membrane depolarization, and depolarization arrested growth independently of shock, indicating that electrical signals implement the negative feedback characteristic of homeostasis. Thus, competing influences of membrane tension and cell-wall mechanical stress on growth allow cells to rapidly correct for mismatches between membrane and wall synthesis rates, ensuring balanced growth.
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http://dx.doi.org/10.1016/j.cels.2017.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985661PMC
December 2017

Cytoplasmic Flow and Mixing Due to Deformation of Motile Cells.

Biophys J 2017 Nov;113(9):2077-2087

Department of Biochemistry, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California; Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California.

The cytoplasm of a living cell is a dynamic environment through which intracellular components must move and mix. In motile, rapidly deforming cells such as human neutrophils, bulk cytoplasmic flow couples cell deformation to the transport and dispersion of cytoplasmic particles. Using particle-tracking measurements in live neutrophil-like cells, we demonstrate that fluid flow associated with the cell deformation contributes to the motion of small acidic organelles, dominating over diffusion on timescales above a few seconds. We then use a general physical model of particle dispersion in a deforming fluid domain to show that transport of organelle-sized particles between the cell periphery and the bulk can be enhanced by dynamic deformation comparable to that observed in neutrophils. Our results implicate an important mechanism contributing to organelle transport in these motile cells: cytoplasmic flow driven by cell shape deformation.
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http://dx.doi.org/10.1016/j.bpj.2017.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685780PMC
November 2017

Adhesion to the host cell surface is sufficient to mediate entry into epithelial cells.

Mol Biol Cell 2017 Nov 6;28(22):2945-2957. Epub 2017 Sep 6.

Department of Biochemistry, Stanford University, Stanford, CA 94305

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen during an in vivo infection. binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.
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http://dx.doi.org/10.1091/mbc.E16-12-0851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662255PMC
November 2017

Non-model model organisms.

BMC Biol 2017 06 29;15(1):55. Epub 2017 Jun 29.

Department of Genetics, Stanford University, Stanford, CA, 94305, USA.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.
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http://dx.doi.org/10.1186/s12915-017-0391-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492503PMC
June 2017

Visualization of mycobacterial membrane dynamics in live cells.

J Am Chem Soc 2017 03 23;139(9):3488-3495. Epub 2017 Feb 23.

Department of Chemistry, Stanford University , Stanford, California 94305, United States.

Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose-fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein-trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.
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http://dx.doi.org/10.1021/jacs.6b12541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345120PMC
March 2017

Adhesion-Dependent Wave Generation in Crawling Cells.

Curr Biol 2017 Jan 8;27(1):27-38. Epub 2016 Dec 8.

Courant Institute and Department of Biology, New York University, New York, NY 10012, USA. Electronic address:

Dynamic actin networks are excitable. In migrating cells, feedback loops can amplify stochastic fluctuations in actin dynamics, often resulting in traveling waves of protrusion. The precise contributions of various molecular and mechanical interactions to wave generation have been difficult to disentangle, in part due to complex cellular morphodynamics. Here we used a relatively simple cell type-the fish epithelial keratocyte-to define a set of mechanochemical feedback loops underlying actin network excitability and wave generation. Although keratocytes are normally characterized by the persistent protrusion of a broad leading edge, increasing cell-substrate adhesion strength results in waving protrusion of a short leading edge. We show that protrusion waves are due to fluctuations in actin polymerization rates and that overexpression of VASP, an actin anti-capping protein that promotes actin polymerization, switches highly adherent keratocytes from waving to persistent protrusion. Moreover, VASP localizes both to adhesion complexes and to the leading edge. Based on these results, we developed a mathematical model for protrusion waves in which local depletion of VASP from the leading edge by adhesions-along with lateral propagation of protrusion due to the branched architecture of the actin network and negative mechanical feedback from the cell membrane-results in regular protrusion waves. Consistent with our model simulations, we show that VASP localization at the leading edge oscillates, with VASP leading-edge enrichment greatest just prior to protrusion initiation. We propose that the mechanochemical feedbacks underlying wave generation in keratocytes may constitute a general module for establishing excitable actin dynamics in other cellular contexts.
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http://dx.doi.org/10.1016/j.cub.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225140PMC
January 2017

Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.

Cell 2016 Oct;167(3):670-683.e10

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.
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http://dx.doi.org/10.1016/j.cell.2016.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097866PMC
October 2016

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria.

mBio 2016 08 30;7(4). Epub 2016 Aug 30.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA

Unlabelled: Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae

Importance: Much of our knowledge on bacterial cytokinesis comes from studying rod-shaped model organisms such as Escherichia coli and Bacillus subtilis Less is known about variations in this process among different bacterial species. While cell division in many bacteria has been characterized to some extent genetically or biochemically, few species have been examined using video microscopy to uncover the kinetics of cytokinesis and daughter cell separation (DCS). In this work, we found that fast (millisecond) DCS is exhibited by species in two independent clades of Gram-positive bacteria and is particularly prevalent among the Actinobacteria, a diverse group that includes significant pathogens as well as bacteria that generate medically important antibiotics.
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http://dx.doi.org/10.1128/mBio.00952-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999543PMC
August 2016

Relative Rates of Surface and Volume Synthesis Set Bacterial Cell Size.

Cell 2016 Jun;165(6):1479-1492

Biophysics Program, Department of Biochemistry and Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA. Electronic address:

Many studies have focused on the mechanisms underlying length and width determination in rod-shaped bacteria. Here, we focus instead on cell surface area to volume ratio (SA/V) and demonstrate that SA/V homeostasis underlies size determination. We propose a model whereby the instantaneous rates of surface and volume synthesis both scale with volume. This model predicts that these relative rates dictate SA/V and that cells approach a new steady-state SA/V exponentially, with a decay constant equal to the volume growth rate. To test this, we exposed diverse bacterial species to sublethal concentrations of a cell wall biosynthesis inhibitor and observed dose-dependent decreases in SA/V. Furthermore, this decrease was exponential and had the expected decay constant. The model also quantitatively describes SA/V alterations induced by other chemical, nutritional, and genetic perturbations. We additionally present evidence for a surface material accumulation threshold underlying division, sensitizing cell length to changes in SA/V requirements.
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http://dx.doi.org/10.1016/j.cell.2016.05.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933963PMC
June 2016