Mechanisms Controlling Cell Size and Shape during Isotropic Cell Spreading

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.     

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.     

Calpain Regulates Actin Remodeling during Cell Spreading

Previous studies suggest that the Ca²⁺-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading.     

Hybrid Spreading Mechanisms and T Cell Activation Shape the Dynamics of HIV-1 Infection

HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments’ influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies.     

Cell Blebbing and Membrane Area Homeostasis in Spreading and Retracting Cells

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.     

Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading

Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments – spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1) During early spreading, cells form initial contacts with the surface. 2) The middle spreading phase exhibits rapidly increasing attachment area. 3) Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters – a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules that, together, determine the overall motility function. Our data and algorithms are publicly available to encourage further exploration.     

The Role of Cell Volume in the Dynamics of Seizure,Spreading Depression,and Anoxic Depolarization

Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little of how cell volume affects neuronal dynamics. We here performed the first detailed study of the effects of cell volume on neuronal dynamics. By incorporating cell swelling together with dynamic ion concentrations and oxygen supply into Hodgkin-Huxley type spiking dynamics, we demonstrate the spontaneous transition between epileptic seizure and spreading depression states as the cell swells and contracts in response to changes in osmotic pressure. Our use of volume as an order parameter further revealed a dynamical definition for the experimentally described physiological ceiling that separates seizure from spreading depression, as well as predicted a second ceiling that demarcates spreading depression from anoxic depolarization. Our model highlights the neuroprotective role of glial K buffering against seizures and spreading depression, and provides novel insights into anoxic depolarization and the relevant cell swelling during ischemia. We argue that the dynamics of seizures, spreading depression, and anoxic depolarization lie along a continuum of the repertoire of the neuron membrane that can be understood only when the dynamic ion concentrations, oxygen homeostasis,and cell swelling in response to osmotic pressure are taken into consideration. Our results demonstrate the feasibility of a unified framework for a wide range of neuronal behaviors that may be of substantial importance in the understanding of and potentially developing universal intervention strategies for these pathological states.     

Cell Surface Topography Is a Regulator of Molecular Interactions during Chemokine-Induced Neutrophil Spreading

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (α_Lβ₂) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.     

Cell Surface Topography Is a Regulator of Molecular Interactions during Chemokine-Induced Neutrophil Spreading

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (α_Lβ₂) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.     

Red Blood Cell Membrane Dynamics during Malaria Parasite Egress

Precisely how malaria parasites exit from infected red blood cells to further spread the disease remains poorly understood. It has been shown recently, however, that these parasites exploit the elasticity of the cell membrane to enable their egress. Based on this work, showing that parasites modify the membrane’s spontaneous curvature, initiating pore opening and outward membrane curling, we develop a model of the dynamics of the red blood cell membrane leading to complete parasite egress. As a result of the three-dimensional, axisymmetric nature of the problem, we find that the membrane dynamics involve two modes of elastic-energy release: 1), at short times after pore opening, the free edge of the membrane curls into a toroidal rim attached to a membrane cap of roughly fixed radius; and 2), at longer times, the rim radius is fixed, and lipids in the cap flow into the rim. We compare our model with the experimental data of Abkarian and co-workers and obtain an estimate of the induced spontaneous curvature and the membrane viscosity, which control the timescale of parasite release. Finally, eversion of the membrane cap, which liberates the remaining parasites, is driven by the spontaneous curvature and is found to be associated with a breaking of the axisymmetry of the membrane.     

Live Cell Interferometry Quantifies Dynamics of Biomass Partitioning during Cytokinesis

The equal partitioning of cell mass between daughters is the usual and expected outcome of cytokinesis for self-renewing cells. However, most studies of partitioning during cell division have focused on daughter cell shape symmetry or segregation of chromosomes. Here, we use live cell interferometry (LCI) to quantify the partitioning of daughter cell mass during and following cytokinesis. We use adherent and non-adherent mouse fibroblast and mouse and human lymphocyte cell lines as models and show that, on average, mass asymmetries present at the time of cleavage furrow formation persist through cytokinesis. The addition of multiple cytoskeleton-disrupting agents leads to increased asymmetry in mass partitioning which suggests the absence of active mass partitioning mechanisms after cleavage furrow positioning.     

Dynamics of Defense Responses and Cell Fate Change during Arabidopsis-Pseudomonas syringae Interactions

Plant-pathogen interactions involve sophisticated action and counteraction strategies from both parties. Plants can recognize pathogen derived molecules, such as conserved pathogen associated molecular patterns (PAMPs) and effector proteins, and subsequently activate PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, pathogens can evade such recognitions and suppress host immunity with effectors, causing effector-triggered susceptibility (ETS). The differences among PTI, ETS, and ETI have not been completely understood. Toward a better understanding of PTI, ETS, and ETI, we systematically examined various defense-related phenotypes of Arabidopsis infected with different Pseudomonas syringae pv. maculicola ES4326 strains, using the virulence strain DG3 to induce ETS, the avirulence strain DG34 that expresses avrRpm1 (recognized by the resistance protein RPM1) to induce ETI, and HrcC^- that lacks the type three secretion system to activate PTI. We found that plants infected with different strains displayed dynamic differences in the accumulation of the defense signaling molecule salicylic acid, expression of the defense marker gene PR1, cell death formation, and accumulation/localization of the reactive oxygen species, H₂O₂. The differences between PTI, ETS, and ETI are dependent on the doses of the strains used. These data support the quantitative nature of PTI, ETS, and ETI and they also reveal qualitative differences between PTI, ETS, and ETI. Interestingly, we observed the induction of large cells in the infected leaves, most obviously with HrcC^- at later infection stages. The enlarged cells have increased DNA content, suggesting a possible activation of endoreplication. Consistent with strong induction of abnormal cell growth by HrcC^-, we found that the PTI elicitor flg22 also activates abnormal cell growth, depending on a functional flg22-receptor FLS2. Thus, our study has revealed a comprehensive picture of dynamic changes of defense phenotypes and cell fate determination during Arabidopsis-P. syringae interactions, contributing to a better understanding of plant defense mechanisms.     

Alterations in Peripheral Blood B Cell Subsets and Dynamics of B Cell Responses during Human Schistosomiasis

Antibody responses are thought to play an important role in control of Schistosoma infections, yet little is known about the phenotype and function of B cells in human schistosomiasis. We set out to characterize B cell subsets and B cell responses to B cell receptor and Toll-like receptor 9 stimulation in Gabonese schoolchildren with Schistosoma haematobium infection. Frequencies of memory B cell (MBC) subsets were increased, whereas naive B cell frequencies were reduced in the schistosome-infected group. At the functional level, isolated B cells from schistosome-infected children showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production. Importantly, 6-months after 3 rounds of praziquantel treatment, frequencies of naive B cells were increased, MBC frequencies were decreased and with the exception of TNF-α production, B cell responsiveness was restored to what was seen in uninfected children. These data show that S. haematobium infection leads to significant changes in the B cell compartment, both at the phenotypic and functional level.     

srGAP2 Arginine Methylation Regulates Cell Migration and Cell Spreading through Promoting Dimerization

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225–538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.     

Spreading of Salmonellas during Cattle Slaughtering

The spread of salmonellas during the slaughtering of cattle in the Berlin (West) slaughterhouse was investigated. The slaughterline was divided into 12 areas. During the first examination, which lasted eight months, the equipment and implements at each stage of slaughter, and faeces of cattle were tested. Salmonellas were not isolated from any of the 226 samples of faeces whereas 14 isolations (1.8%) were made from the 768 swabs taken from equipment. Most isolates were obtained within the step of opening the abdominal cavity.
In a second survey 61 (4.3%) of 1392 swab samples and 2 (0.75%) of 267 faeces samples were positive. Of the different processing steps, cutting off the hooves and loosening the skin of legs gave the highest recovery of salmonellas. Moderate recoveries were obtained after opening the abdominal cavity. Removal of head, loosening the skin of head, removing the hide and splitting the breastbone and the carcass did not result in the isolation of the organism. Salmonella anatum was the most common isolate. The same serotypes were isolated from both carcasses and equipment.     

Myosin II Motor Proteins with Different Functions Determine the Fate of Lamellipodia Extension during Cell Spreading

Non-muscle cells express multiple myosin-II motor proteins myosin IIA, myosin IIB and myosin IIC transcribed from different loci in the human genome. Due to a significant homology in their sequences, these ubiquitously expressed myosin II motor proteins are believed to have overlapping cellular functions, but the mechanistic details are not elucidated. The present study uncovered a mechanism that coordinates the distinctly localized myosin IIA and myosin IIB with unexpected opposite mechanical roles in maneuvering lamellipodia extension, a critical step in the initiation of cell invasion, spreading, and migration. Myosin IIB motor protein by localizing at the front drives lamellipodia extension during cell spreading. On the other hand, myosin IIA localizes next to myosin IIB and attenuates or retracts lamellipodia extension. Myosin IIA and IIB increase cell adhesion by regulating focal contacts formation in the spreading margins and central part of the spreading cell, respectively. Spreading cells expressing both myosin IIA and myosin IIB motor proteins display an organized actin network consisting of retrograde filaments, arcs and central filaments attached to focal contacts. This organized actin network especially arcs and focal contacts formation in the spreading margins were lost in myosin IIÂ cells. Surprisingly, myosin II$\widehat{B}$ cells displayed long parallel actin filaments connected to focal contacts in the spreading margins. Thus, with different roles in the regulation of the actin network and focal contacts formation, both myosin IIA and IIB determine the fate of lamellipodia extension during cell spreading.     

Role of Actin Filaments in Correlating Nuclear Shape and Cell Spreading

It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus.     

Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.