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Micah Dembo

Publications and source records attributed to Micah Dembo.

18 recordsLinked to original sources
PubMed2007

Neutrophil traction stresses are concentrated in the uropod during migration.

We find that in contrast to strongly adherent, slow moving cells such as fibroblasts, neutrophils exert contractile stresses largely in the rear of the cell (uropod) relative to the direction of motion. Rather than the leading edge pulling the cell, the rear is both anchoring the cell and the area in which the contractile forces are concentrated. These tractions rapidly reorient themselves during a turn, on a timescale of seconds to minutes, and their repositioning precedes and sets the direction of motion during a turn. We find the total average root mean-squared traction force to be 28+/-10 nN during chemokinesis, and 67+/-10 nN during chemotaxis. We hypothesize that the contraction forces in the back of the neutrophil not only break uropodial adhesive contacts but also create a rearward squeezing contractility, as seen in amoeboid or amoeboidlike cells and the formation of blebs in cells, causing a flow of intracellular material to the fluidlike lamellipod. Our findings suggest an entirely new model of neutrophil locomotion.

Cell Adhesion↗
PubMed2006

Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase.

Adhesive cells show complex mechanical interactions with the substrate, however the exact mechanism of such interactions, termed traction forces, is still unclear. To address this question we have measured traction forces of fibroblasts treated with agents that affect the myosin II-dependent contractile mechanism. Using the potent myosin II inhibitor blebbistatin, we demonstrate that traction forces are strongly dependent on a functional myosin II heavy chain. Since myosin II is regulated by both the myosin light chain kinase (MLCK) and, directly or indirectly, the Rho-associated kinase (ROCK), we examined the effects of inhibitors against these kinases. Interestingly, inhibition of the myosin light chain kinase had no detectable effect, while inhibition of the Rho-dependent kinase caused strong inhibition of traction forces. Our results indicate that ROCK and MLCK play non-redundant roles in regulating myosin II functions, and that a subset of myosin II, regulated by the Rho small GTPase, may be responsible for the regulation of traction forces in migrating fibroblasts.

Animals↗
PubMed2006

Modulation of cell adhesion and motility in the immune system by Myo1f.

Although class I myosins are known to play a wide range of roles, the physiological function of long-tailed class I myosins in vertebrates remains elusive. We demonstrated that one of these proteins, Myo1f, is expressed predominantly in the mammalian immune system. Cells from Myo1f-deficient mice exhibited abnormally increased adhesion and reduced motility, resulting from augmented exocytosis of beta2 integrin-containing granules. Also, the cortical actin that co-localizes with Myo1f was reduced in Myo1f-deficient cells. In vivo, Myo1f-deficient mice showed increased susceptibility to infection by Listeria monocytogenes and an impaired neutrophil response. Thus, Myo1f directs immune cell motility and innate host defense against infection.

Actins↗
PubMed2006

Mechanics of neutrophil phagocytosis: experiments and quantitative models.

To quantitatively characterize the mechanical processes that drive phagocytosis, we observed the FcgammaR-driven engulfment of antibody-coated beads of diameters 3 mum to 11 mum by initially spherical neutrophils. In particular, the time course of cell morphology, of bead motion and of cortical tension were determined. Here, we introduce a number of mechanistic models for phagocytosis and test their validity by comparing the experimental data with finite element computations for multiple bead sizes. We find that the optimal models involve two key mechanical interactions: a repulsion or pressure between cytoskeleton and free membrane that drives protrusion, and an attraction between cytoskeleton and membrane newly adherent to the bead that flattens the cell into a thin lamella. Other models such as cytoskeletal expansion or swelling appear to be ruled out as main drivers of phagocytosis because of the characteristics of bead motion during engulfment. We finally show that the protrusive force necessary for the engulfment of large beads points towards storage of strain energy in the cytoskeleton over a large distance from the leading edge ( approximately 0.5 microm), and that the flattening force can plausibly be generated by the known concentrations of unconventional myosins at the leading edge.

Animals↗
PubMed2005

The dynamics and mechanics of endothelial cell spreading.

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.

Actins↗
PubMed2005

Mechanics of neutrophil phagocytosis: behavior of the cortical tension.

The mechanical implementation of phagocytosis requires a well-coordinated deployment of cytoplasm and membrane during the creation of a phagosome. We follow the time course of this process in initially round passive neutrophils presented with antibody-coated beads of radii 1.1 to 5.5 microm. In particular, we monitor the cortical tension as the apparent cellular surface area increases due to cell-driven deformations induced by phagocytosis. The behavior of the tension is then compared with conditions of similar area expansion caused by externally imposed deformations during cell aspiration into a micropipette. Whereas the resting tension remains low for an area expansion of up to only 30% during aspiration, it remains low even after an area expansion of up to 80% in phagocytosis. This is probably the result of membrane insertion from inner stores by exocytosis. We further find that the onset of viscous tension, proportional to the rate of area expansion and caused by the unfurling of plasma membrane wrinkles, is significantly delayed in phagocytosis compared with aspiration. We propose that this is the result of phagocytosis-triggered enzymatic activity that releases spare plasma membrane normally sequestered by velcro-like bonds in a reservoir of surface folds and villi.

Biophysics↗
PubMed2005

Tensional homeostasis and the malignant phenotype.

Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.

3T3 Cells↗
PubMed2004

Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors.

Fibroblasts in 2D cultures differ dramatically in behavior from those in the 3D environment of a multicellular organism. However, the basis of this disparity is unknown. A key difference is the spatial arrangement of anchored extracellular matrix (ECM) receptors to the ventral surface in 2D cultures and throughout the entire surface in 3D cultures. Therefore, we asked whether changing the topography of ECM receptor anchorage alone could invoke a morphological response. By using polyacrylamide-based substrates to present anchored fibronectin or collagen on dorsal cell surfaces, we found that well spread fibroblasts in 2D cultures quickly changed into a bipolar or stellate morphology similar to fibroblasts in vivo. Cells in this environment lacked lamellipodia and large actin bundles and formed small focal adhesions only near focused sites of protrusion. These responses depend on substrate rigidity, calcium ion, and, likely, the calcium-dependent protease calpain. We suggest that fibroblasts respond to both spatial distribution and mechanical input of anchored ECM receptors. Changes in cell shape may in turn affect diverse cellular activities, including gene expression, growth, and differentiation, as shown in numerous previous studies.

Acrylic Resins↗
PubMed2004

Dynamic changes in traction forces with DC electric field in osteoblast-like cells.

Primary bovine osteoblasts and human osteosarcoma cells exposed to direct-current electric fields undergo processes of retraction and elongation ultimately resulting in the realignment of the long cellular axis perpendicular to the electric field. The time taken for this reorientation was inversely correlated to field strength within a certain range. Cellular force output during reorientation was analyzed using a simple modification of traction force microscopy. The first detectable reaction was an increase in average traction force magnitude occurring between 10 and 30 seconds of electric field exposure. In the following 2 to 15 minutes traction forces at margins tangential to the electric field decreased below their initial values. Phase-contrast microscopy revealed elongating protrusions at these margins several minutes later. We could not correlate the initial traction changes with any change in intracellular free calcium levels measured using the fluorescent dye Fura-2 AM.

Acrylic Resins↗
PubMed2004

Equilibrium thermodynamics of cell-cell adhesion mediated by multiple ligand-receptor pairs.

In many situations, cell-cell adhesion is mediated by multiple ligand-receptor pairs. For example, the interaction between T cells and antigen-presenting cells of the immune system is mediated not only by T cell receptors and their ligands (peptide-major histocompatibility complex) but also by binding of intracellular adhesion molecules. Interestingly, these binding pairs have different resting lengths. Fluorescent labeling reveals segregation of the longer adhesion molecules from the shorter T cell receptors in this case. Here, we explore the thermal equilibrium of a general cell-cell interaction mediated by two ligand-receptor pairs to examine competition between the elasticity of the cell wall, nonspecific intercellular repulsion, and bond formation, leading to segregation of bonds of different lengths at equilibrium. We make detailed predictions concerning the relationship between physical properties of the membrane and ligand-receptor pairs and equilibrium pattern formation, and suggest experiments to refine our understanding of the system. We demonstrate our model by application to the T cell/antigen-presenting-cell system and outline applications to natural killer cell adhesion.

Binding Sites↗
PubMed2004

Rho mediates the shear-enhancement of endothelial cell migration and traction force generation.

The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK.

Animals↗
PubMed2003

Nonmuscle myosin IIb is involved in the guidance of fibroblast migration.

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.

Acrylic Resins↗
PubMed2003

Demonstration of altered fibroblast contractile activity in hypertensive heart disease.

OBJECTIVE: The aim of this study is to investigate the idea that altered fibroblast contractile activity is involved in the pathogenesis of hypertensive heart disease (HHD). METHODS: Cell area and contraction are quantified using the traction force microscopy technique for cardiac fibroblasts isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. RESULTS: The data indicate that there are marked phenotypic differences between the two cell types. For instance, WKY fibroblasts exert an average traction stress of approximately 3.3 kPa and have an area of approximately 2640 microm(2). Under identical conditions the SHR fibroblasts have an area approximately 1.45 times larger (p<0.01) and exert an average stress approximately 1.86 times higher (p<0.01). Challenging WKY fibroblasts with 1 micromol/l angiotensin II (Ang II) gradually causes a approximately 2-fold increase in traction after 1 h while simultaneously causing a approximately 28% decrease in area. In contrast, Ang II has no effect on SHR fibroblasts. The data also show that WKY and SHR cells respond in different ways when challenged with irbesartan (Irb). The addition of 1 micromol/l Irb initially causes WKY cells to decrease their average traction output by approximately 50% after approximately 10 min. Subsequently, contractile activity begins to recover and returns to normal after 1 h. The SHR cells also decrease their tractions by approximately 50%, but this decrease requires 30 min for completion and there is no recovery to the initial contractile state. For both cell types, Irb produces no significant effect on area and the combined effect of equimolar Irb and Ang II is the same as Irb alone. CONCLUSION: These in vitro data suggest that among the many factors producing hypertensive heart disease in SHR's are excessive contraction of their cardiac fibroblasts and defective control of fibroblast contraction by Ang II.

Animals↗
PubMed2003

Regulation of mechanical interactions between fibroblasts and the substratum by stretch-activated Ca2+ entry.

Ca2+ ions have long been implicated in regulating various aspects of cell movements. We found that stretching forces applied through flexible substrata induced increases in both intracellular Ca2+ concentration and traction forces of NIH3T3 fibroblasts. Conversely, application of gadolinium, an inhibitor of stretch-activated ion channels, or removal of extracellular free Ca2+ caused inhibition of traction forces. Gadolinium treatment also inhibited cell migration without affecting the spread morphology or protrusive activities. Local application of gadolinium to the trailing region had no detectable effect on the overall traction forces, while local application to the leading edge caused a global inhibition of traction forces and cell migration, suggesting that stretch-activated channels function primarily at the leading edge. Immunofluorescence microscopy indicated that gadolinium caused a pronounced decrease in vinculin and phosphotyrosine concentrations at focal adhesions. Our observations suggest that stretch-activated Ca2+ entry in the frontal region regulates the organization of focal adhesions and the output of mechanical forces. This mechanism probably plays an important role in sustaining cell migration and in mediating active and passive responses to mechanical signals.

Animals↗
PubMed2003

The mechanics of neutrophils: synthetic modeling of three experiments.

Much experimental data exist on the mechanical properties of neutrophils, but so far, they have mostly been approached within the framework of liquid droplet models. This has two main drawbacks: 1), It treats the cytoplasm as a single phase when in reality, it is a composite of cytosol and cytoskeleton; and 2), It does not address the problem of active neutrophil deformation and force generation. To fill these lacunae, we develop here a comprehensive continuum-mechanical paradigm of the neutrophil that includes proper treatment of the membrane, cytosol, and cytoskeleton components. We further introduce two models of active force production: a cytoskeletal swelling force and a polymerization force. Armed with these tools, we present computer simulations of three classic experiments: the passive aspiration of a neutrophil into a micropipette, the active extension of a pseudopod by a neutrophil exposed to a local stimulus, and the crawling of a neutrophil inside a micropipette toward a chemoattractant against a varying counterpressure. Principal results include: 1), Membrane cortical tension is a global property of the neutrophil that is affected by local area-increasing shape changes. We argue that there exists an area dilation viscosity caused by the work of unfurling membrane-storing wrinkles and that this viscosity is responsible for much of the regulation of neutrophil deformation. 2), If there is no swelling force of the cytoskeleton, then it must be endowed with a strong cohesive elasticity to prevent phase separation from the cytosol during vigorous suction into a capillary tube. 3), We find that both swelling and polymerization force models are able to provide a unifying fit to the experimental data for the three experiments. However, force production required in the polymerization model is beyond what is expected from a simple short-range Brownian ratchet model. 4), It appears that, in the crawling of neutrophils or other amoeboid cells inside a micropipette, measurement of velocity versus counterpressure curves could provide a determination of whether cytoskeleton-to-cytoskeleton interactions (such as swelling) or cytoskeleton-to-membrane interactions (such as polymerization force) are predominantly responsible for active protrusion.

Cell Membrane↗
PubMed2003

Influence of type I collagen surface density on fibroblast spreading, motility, and contractility.

We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low.

Animals↗
PubMed2003

Measurements of cell-generated deformations on flexible substrata using correlation-based optical flow.

The optical flow algorithm presented here is a robust method that rapidly yields a high-density field of substrate displacement vectors based on two optical images. We found that one of the limiting factors, at least for inexperienced experimentalists, is the consistency of focusing or the drift in microscope focus. However, with properly collected images the standard error of the measurement was estimated to be on the order of +/- 0.10 pixels. Finally, although the discussion has been focused on the displacement of flexible substrata, a similar method should be applicable for detecting movements on other types of images, as long as the movement involves a certain degree of local coordination.

Acrylic Resins↗