Membrane dynamics correlate with formation of signaling clusters during cell spreading

The morphology and duration of contacts between cells and adhesive surfaces play a key role in several biological processes, such as cell migration, cell differentiation, and the immune response. The interaction of receptors on the cell membrane with ligands on the adhesive surface leads to triggering of signaling pathways, which allow cytoskeletal rearrangement, and large-scale deformation of the cell membrane, which allows the cell to spread over the substrate. Despite numerous studies of cell spreading, the nanometer-scale dynamics of the membrane during formation of contacts, spreading, and initiation of signaling are not well understood. Using interference reflection microscopy, we study the kinetics of cell spreading at the micron scale, as well as the topography and fluctuations of the membrane at the nanometer scale during spreading of Jurkat T cells on antibody-coated substrates. We observed two modes of spreading, which were characterized by dramatic differences in membrane dynamics and topography. Formation of signaling clusters was closely related to the movement and morphology of the membrane in contact with the activating surface. Our results suggest that cell membrane morphology may be a critical constraint on signaling at the cell-substrate interface.     

Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces

Complex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.     

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.     

Interaction and fusion dynamics between cellular blebs

Membrane blebbing, as a mechanism for cells to regulate their internal pressure and membrane tension, is believed to play important roles in processes such as cell migration, spreading and apoptosis. However, the fundamental question of how different blebs interact with each other during their life cycles remains largely unclear. Here, we report a combined theoretical and experimental investigation to examine how the growth and retraction of a cellular bleb are influenced by neighboring blebs as well as the fusion dynamics between them. Specifically, a boundary integral model was developed to describe the shape evolution of cell membrane during the blebbing/retracting process. We showed that a drop in the intracellular pressure will be induced by the formation of a bleb whose retraction then restores the pressure level. Consequently, the volume that a second bleb can reach was predicted to heavily depend on its initial weakened size and the time lag with respect to the first bleb, all in quantitative agreement with our experimental observations. In addition, it was found that as the strength of membrane-cortex adhesion increases, the possible coalescence of two neighboring blebs changes from smooth fusion to abrupt coalescence and eventually to no fusion at all. Phase diagrams summarizing the dependence of such transition on key physical factors, such as the intracellular pressure and bleb separation, were also obtained.     

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).     

A mathematical model of the coupled mechanisms of cell adhesion,contraction and spreading

Recent research has shown that cell spreading is highly dependent on the contractility of its cytoskeleton and the mechanical properties of the environment it is located in. The dynamics of such process is critical for the development of tissue engineering strategy but is also a key player in wound contraction, tissue maintenance and angiogenesis. To better understand the underlying physics of such phenomena, the paper describes a mathematical formulation of cell spreading and contraction that couples the processes of stress fiber formation, protrusion growth through actin polymerization at the cell edge and dynamics of cross-membrane protein (integrins) enabling cell-substrate attachment. The evolving cell’s cytoskeleton is modeled as a mixture of fluid, proteins and filaments that can exchange mass and generate contraction. In particular, besides self-assembling into stress fibers, actin monomers able to polymerize into an actin meshwork at the cell’s boundary in order to push the membrane forward and generate protrusion. These processes are possible via the development of cell-substrate attachment complexes that arise from the mechano-sensitive equilibrium of membrane proteins, known as integrins. After deriving the governing equation driving the dynamics of cell evolution and spreading, we introduce a numerical solution based on the extended finite element method, combined with a level set formulation. Numerical simulations show that the proposed model is able to capture the dependency of cell spreading and contraction on substrate stiffness and chemistry. The very good agreement between model predictions and experimental observations suggests that mechanics plays a strong role into the coupled mechanisms of contraction, adhesion and spreading of adherent cells.     

Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

Dynamic regulation of the actin cytoskeleton is important for cell motility, spreading and the formation of membrane surface extensions such as lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In the present study, the live-cell morphology of calpain-knockout and wild-type cells was examined by time-lapse fluorescence microscopy, and a role of calpain in mediating the formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels of tropomyosin in calpain-knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dynamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild-type cells. These results suggest a regulatory connection between calpain and RhoGDI-1 in promoting formation of membrane blebs.     

The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.     

The effects of cell-cell contact on the spreading of pigmented retina epithelial cells in culture

The behaviour of chick embryo pigmented retina epithelial (PRE) cells has been studied in living and fixed cultures. Isolated PRE cells lacking contacts with other cells were characteristically only poorly spread upon the substrate, blebbed vigorously and lacked leading lamellae. PRE cells incorporated into islands or sheets of cells were extensively spread upon the substrate, lacked blebs and displayed typical leading lamellae if marginally positioned in an island. Observations of living cultures demonstrated that within 3 h of establishing contact with an island of cells a previously isolated PRE cell lost the morphology characteristic of isolated cells and became indistinguishable from its neighbours in the island. Measurements of the area of substrate occupied by single cells and cells in 2-cell islands suggests that similar changes occur as two cells make contact to form a 2-cell island. The evidence suggests that these changes are a direct response to the establishment of a cell-cell contact and I propose that the phenomenon be termed ‘contact-induced spreading’.Contact-induced spreading is not an ‘all or none’ phenomenon since isolated PRE cells can spread extensively and cease blebbing in the absence of cell contact. However a given isolated PRE cell spends only a very small proportion of its time displaying this well spread morphology and therefore at any time the majority of isolated PRE cells display the poorly spread morphology.The possible relationship between contact-induced spreading and other cellular interactions known to be dependent on cell-cell contact is discussed.     

Cytoarchitecture of ras oncogene-expressing tumor cells: butyrate modulation of substrate adhesion, cytoskeletal actin content and subcellular microfilament distribution

1. The subcellular distribution of particular cytoskeletal (CSK) and cell-substrate adhesive elements was assessed during the morphologic response of cultured tumor cells to the shape modulating agent sodium butyrate (NaB). 2. NaB induced marked increases in cellular and CSK actin content and in the matrix-associated proteins fibronectin and p52. 3. Subcellular fractionation indicated disproportionate increases in the actin content of the substrate-attached cellular residue (SAM fraction) which contains the majority of cell-substrate adhesive elements. 4. Augmented cell spreading and substrate attachment characteristic of NaB-treated cells is likely due to increased elaboration of cell-to-substrate adhesive structures and reflected in an enhanced deposition of actin into the CSK and SAM compartments.     

Relation between cell activity and the distribution of cytoplasmic actin and myosin

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.     

pH sensing by FAK-His58 regulates focal adhesion remodeling

Intracellular pH (pHi) dynamics regulates diverse cellular processes, including remodeling of focal adhesions. We now report that focal adhesion kinase (FAK), a key regulator of focal adhesion remodeling, is a pH sensor responding to physiological changes in pH. The initial step in FAK activation is autophosphorylation of Tyr397, which increased with higher pHi. We used a genetically encoded biosensor to show increased pH at focal adhesions as they mature during cell spreading. We also show that cells with reduced pHi had attenuated FAK-pY397 as well as defective cell spreading and focal adhesions. Mutagenesis studies indicated FAK-His58 is critical for pH sensing and molecular dynamics simulations suggested a model in which His58 deprotonation drives conformational changes that may modulate accessibility of Tyr397 for autophosphorylation. Expression of FAK-H58A in fibroblasts was sufficient to restore defective autophosphorylation and cell spreading at low pHi. These data are relevant to understanding cancer metastasis, which is dependent on increased pHi and FAK activity.     

Real-time monitoring of epithelial cell-cell and cell-substrate interactions by infrared surface plasmon spectroscopy

The development of novel technologies capable of monitoring the dynamics of cell-cell and cell-substrate interactions in real time and a label-free manner is vital for gaining deeper insights into these most fundamental cellular processes. However, the label-free technologies available today provide only limited information on these processes. Here, we report a new (to our knowledge) infrared surface plasmon resonance (SPR)-based methodology that can resolve distinct phases of cell-cell and cell-substrate adhesion of polarized Madin Darby canine kidney epithelial cells. Due to the extended penetration depth of the infrared SP wave, the dynamics of cell adhesion can be detected with high accuracy and high temporal resolution. Analysis of the temporal variation of the SPR reflectivity spectrum revealed the existence of multiple phases in epithelial cell adhesion: initial contact of the cells with the substrate (cell deposition), cell spreading, formation of intercellular contacts, and subsequent generation of cell clusters. The final formation of a continuous cell monolayer could also be sensed. The SPR measurements were validated by optical microscopy imaging. However, in contrast to the SPR method, the optical analyses were laborious and less quantitative, and hence provided only limited information on the dynamics and phases of cell adhesion.     

Nanometer analysis of cell spreading on matrix-coated surfaces reveals two distinct cell states and STEPs

When mouse embryonic fibroblasts in suspension contact a matrix-coated surface, they rapidly adhere and spread. Using total internal reflection fluorescence microscopy of dye-loaded fibroblasts to quantify cell-substrate contact, we found that increasing the surface matrix density resulted in faster spreading initiation whereas lamellipodial dynamics during spreading were unaltered. After spreading initiation, most cells spread in an anisotropic manner through stochastic, transient extension periods (STEPs) with approximately 30 STEPs over 10 min to reach an area of 1300 micro m(2) +/- 300 micro m(2). A second mode of spreading, increased in serum-deprived cells, lacked STEPs and spread in a rapid, isotropic manner for 1-4 min. This isotropic mode was characterized by a high rate of area increase, 340 micro m(2)/min with 78% of the cell edge extending. Anisotropic cells spread slower via STEPs, 126 micro m(2)/min with 34% of the edge extending. During the initial 2-4 min of fast, isotropic spreading, centripetal flow of actin was low (0.8 micro m/min) whereas in anisotropic cells it was high from early times (4.7 micro m/min). After initial isotropic spreading, rearward actin movement increased and isotropic cells displayed STEPs similar to anisotropic cells. Thus, the two cell states display dramatically different spreading whereas long-term motility is based on STEPs.     

Electrical, chemical, and topological addressing of mammalian cells with microfabricated systems

This communication describes our work in electrical, topological, and chemical micromodification of surfaces to modulate cellular form and function. We have addressed the surface physico-chemico-mechano properties of cell culture substrates that play a role in modulating cellular behavior. Single factorial model systems have been built using techniques adapted from microlithography. The tools and techniques of microfabrication, if harnessed and used correctly, can be enabling in elucidating the underlying principles and fundamental forces driving the cell-substrate interface. Additionally, the long-term practical applications of microfabrication in medicine and biomaterial/tissue engineering lie in enabling "communication" with living cells/tissues at the cellular and subcellular levels.     

Actin polymerization and intracellular solvent flow in cell surface blebbing

The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well.     

Attachment of HeLa cells during early G1 phase

Both growth factor directed and integrin dependent signal transduction were shown to take place directly after completion of mitosis. The local activation of these signal transduction cascades was investigated in early G1 cells. Interestingly, various key signal transduction proteins were found in blebs at the cell membrane within 30 min after mitosis. These membrane blebs appeared in round, mitotic-like cells and disappeared rapidly during spreading of the cells in G1 phase. In addition to tyrosine-phosphorylated proteins, the blebs contained also phosphorylated FAK and phosphorylated MAP kinase. The formation of membrane blebs in round, mitotic cells before cell spreading is not specific for mitotic cells, because similar features were observed in trypsinized cells. Just before cell spreading also these cells exhibited membrane blebs containing active signal transduction proteins. Inhibition of signal transduction did not affect membrane bleb formation, suggesting that the membrane blebs were formed independent of signal transduction.     

Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.     

FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling

FilGAP is a newly recognized filamin A (FLNa)-binding RhoGTPase-activating protein. The GTPase-activating protein (GAP) activity of FilGAP is specific for Rac and FLNa binding targets FilGAP to sites of membrane protrusion, where it antagonizes Rac in vivo. Dominant-negative FilGAP constructs lacking GAP activity or knockdown of endogenous FilGAP by small interference RNA (siRNA) induce spontaneous lamellae formation and stimulate cell spreading on fibronectin. Knockdown of endogenous FilGAP abrogates ROCK-dependent suppression of lamellae. Conversely, forced expression of FilGAP induces numerous blebs around the cell periphery and a ROCK-specific inhibitor suppresses bleb formation. ROCK phosphorylates FilGAP, and this phosphorylation stimulates its RacGAP activity and is a requirement for FilGAP-mediated bleb formation. FilGAP is, therefore, a mediator of the well-established antagonism of Rac by RhoA that suppresses leading edge protrusion and promotes cell retraction to achieve cellular polarity.     

Cellular localization of tissue factor in human breast cancer cell lines

Expression of tissue factor (TF), the cellular receptor of clotting factor VII/VIIa, is a feature of certain malignant tumours. The TF gene has been classified as an immediate early gene responsive to serum and cytokines. Thus, the regulation of TF gene expression seems to play a role in cell growth. Recently, we have shown that constitutive TF expression in MCF-7 breast cancer cells is modulated by such growth factors as EGF, TGFα, and IL-1. The present study deals with the immunocytochemically detectable cellular distribution of TF in human breast cancer cell lines MCF-7 and MaTu stimulated by EGF and TGFα. In MCF-7 cells growing logarithmically, stimulation led to a significant increase of TF mRNA after 2 h (in situ hybridization, Northern blot) and to maximum TF expression after 6 h (immunohistochemistry). When decorated by monoclonal antibodies, TF protein showed a pronounced localization at ruffled membrane areas, cell edges, and processes of spreading cells after 6 and 20 h. In more flattened cells TF was concentrated in peripheric lamellae and microspikes communicating with neighbouring cells. After epithelial colony pattern had established, TF was predominantly accumulated at the intercellular boundaries. The vary same distribution patterns as seen in MCF-7 cells were true for the stimulated MaTu cell line. The dynamics and cellular distribution patterns of stimulated TF expression support the hypothesis that TF could be of importance for morphogenic events associated with the growth and differentiation of breast cancer cells in culture.