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Cell states were traditionally defined by how they looked, where they were located, and what functions they performed. In this post-genomic era, the field is largely focused on a molecular view of cell state. Moving forward, we anticipate that the observables used to define cell states will evolve again as single-cell imaging and analytics are advancing at a breakneck pace via the collection of large-scale, systematic cell image datasets and the application of quantitative image-based data science methods. This is, therefore, a key moment in the arc of cell biological research to develop approaches that integrate the spatiotemporal observables of the physical structure and organization of the cell with molecular observables toward the concept of a holistic cell state. In this perspective, we propose a conceptual framework for holistic cell states and state transitions that is data-driven, practical, and useful to enable integrative analyses and modeling across many data types.
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Análise de Célula Única , Humanos , Análise de Célula Única/métodos , AnimaisRESUMO
50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.
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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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Bactérias/crescimento & desenvolvimento , Bactérias/ultraestrutura , Antibacterianos/farmacologia , Fenômenos Biomecânicos , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/crescimento & desenvolvimento , Caulobacter crescentus/ultraestrutura , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/ultraestrutura , Fosfomicina/farmacologia , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/ultraestrutura , Modelos Biológicos , Peptidoglicano , Propriedades de SuperfícieRESUMO
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.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Mecanotransdução Celular , Infecções por Rickettsia/metabolismo , Infecções por Rickettsia/microbiologia , Rickettsia/patogenicidade , Vinculina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Elementos de DNA Transponíveis/genética , Febre/metabolismo , Febre/microbiologia , Humanos , Mutagênese Insercional , Mutação , Rickettsia/metabolismo , alfa Catenina/metabolismoRESUMO
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 understood1. 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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Actinas/química , Actinas/metabolismo , Mitocôndrias/metabolismo , Mitose , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Divisão Celular , Linhagem Celular , Citocinese , Retículo Endoplasmático/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Humanos , Mitocôndrias/química , Neurônios , RatosRESUMO
In a Comment to the Editor, Skóra raises a concern that the modeling framework implemented in Garner et al. (Biophysical Journal, 2023) neglects a potentially important term in the Brownian dynamics simulation of diffusion. Omission of this diffusivity gradient term may lead to an underestimation of the mean and overestimation of the variance of the cytoplasmic viscosity. In this response, we directly address this concern by incorporating this term into our model and showing that for this data set, its effect is negligible and does not alter the conclusions of this work.
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Citoplasma , Difusão , Citoplasma/metabolismo , Viscosidade , Modelos Biológicos , Reologia , NanotecnologiaRESUMO
Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.
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Membrana Celular/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Detergentes/farmacologia , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Suporte de CargaRESUMO
The cytoplasm is a complex, crowded, actively driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40 nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of individual fission yeast (Schizosaccharomyces pombe) cellscells. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak nonergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, the actin and microtubule cytoskeletons, cell-cyle stage, and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40 nm size scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.
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Citoesqueleto , Citoplasma , Difusão , Citosol , Simulação por ComputadorRESUMO
We introduce a framework for end-to-end integrative modeling of 3D single-cell multi-channel fluorescent image data of diverse subcellular structures. We employ stacked conditional ß-variational autoencoders to first learn a latent representation of cell morphology, and then learn a latent representation of subcellular structure localization which is conditioned on the learned cell morphology. Our model is flexible and can be trained on images of arbitrary subcellular structures and at varying degrees of sparsity and reconstruction fidelity. We train our full model on 3D cell image data and explore design trade-offs in the 2D setting. Once trained, our model can be used to predict plausible locations of structures in cells where these structures were not imaged. The trained model can also be used to quantify the variation in the location of subcellular structures by generating plausible instantiations of each structure in arbitrary cell geometries. We apply our trained model to a small drug perturbation screen to demonstrate its applicability to new data. We show how the latent representations of drugged cells differ from unperturbed cells as expected by on-target effects of the drugs.
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Núcleo Celular/fisiologia , Forma Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Espaço Intracelular , Modelos Biológicos , Células Cultivadas , Biologia Computacional , Humanos , Imageamento Tridimensional , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Microscopia de Fluorescência , Análise de Célula ÚnicaRESUMO
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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Miosinas , Pseudópodes , Actinas , Animais , Movimento Celular , CitoesqueletoRESUMO
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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Divisão Celular/fisiologia , Corynebacterium glutamicum/crescimento & desenvolvimento , Mycobacterium smegmatis/crescimento & desenvolvimento , Bactérias , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias , Membrana Celular/metabolismo , Parede Celular/metabolismo , Corynebacterium/crescimento & desenvolvimento , Corynebacterium/metabolismo , Corynebacterium glutamicum/metabolismo , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos , PeptidoglicanoRESUMO
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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Proteínas de Bactérias/metabolismo , Membrana Basal/microbiologia , Listeria monocytogenes/patogenicidade , Proteínas dos Microfilamentos/metabolismo , Complicações Infecciosas na Gravidez/metabolismo , Animais , Feminino , Feto/microbiologia , Humanos , Listeriose/metabolismo , Proteínas de Membrana/metabolismo , Placenta/metabolismo , Placenta/microbiologia , Gravidez , Fatores de Virulência/metabolismoRESUMO
Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that operate differentially in different host cell types.
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Proteínas Fetais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Proteínas Nucleares/metabolismo , Fagocitose/fisiologia , Proteínas/metabolismo , Western Blotting , Células Cultivadas , Citometria de Fluxo , Forminas , Humanos , Listeria monocytogenes , Reação em Cadeia da PolimeraseRESUMO
In this issue of Molecular Cell, Han and Mizuuchi present evidence for a possible Turing-like reaction-diffusion mechanism underlying target immunity by the bacteriophage Mu.
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Cells are dynamic systems capable of spontaneously switching among stable states. One striking example of this is spontaneous symmetry breaking and motility initiation in fish epithelial keratocytes. Although the biochemical and mechanical mechanisms that control steady-state migration in these cells have been well characterized, the mechanisms underlying symmetry breaking are less well understood. In this work, we have combined experimental manipulations of cell-substrate adhesion strength and myosin activity, traction force measurements, and mathematical modeling to develop a comprehensive mechanical model for symmetry breaking and motility initiation in fish epithelial keratocytes. Our results suggest that stochastic fluctuations in adhesion strength and myosin localization drive actin network flow rates in the prospective cell rear above a critical threshold. Above this threshold, high actin flow rates induce a nonlinear switch in adhesion strength, locally switching adhesions from gripping to slipping and further accelerating actin flow in the prospective cell rear, resulting in rear retraction and motility initiation. We further show, both experimentally and with model simulations, that the global levels of adhesion strength and myosin activity control the stability of the stationary state: The frequency of symmetry breaking decreases with increasing adhesion strength and increases with increasing myosin contraction. Thus, the relative strengths of two opposing mechanical forces--contractility and cell-substrate adhesion--determine the likelihood of spontaneous symmetry breaking and motility initiation.
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Movimento Celular , Ciclídeos/metabolismo , Células Epiteliais/citologia , Miosinas/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Adesão Celular , Simulação por Computador , Dinâmica não LinearRESUMO
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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Biologia , Eucariotos , Modelos Animais , Animais , PlantasRESUMO
Front-line tuberculosis (TB) drugs have been characterized extensively inâ vitro and inâ 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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Antituberculosos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Membrana Celular/genética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/metabolismo , Tuberculose/patologiaRESUMO
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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Movimento Celular , Citoplasma/metabolismo , Fenômenos Biomecânicos , Células HL-60 , Humanos , Cinética , Fenômenos Mecânicos , Modelos Biológicos , Neutrófilos/citologiaRESUMO
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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Membrana Celular/metabolismo , Mycobacterium/citologia , Termodinâmica , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Etambutol/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Mycobacterium/efeitos dos fármacosRESUMO
During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (â¼85 min) and the subsequent 11 cycles are short (â¼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.