Intracellular calcium regulates many of the molecular processes that are essential for cell movement. It is required for the production of actomyosin-based contractile forces, the regulation of the structure and dynamics of the actin cytoskeletons, and the formation and disassembly of cell-substratum adhesions. Calcium also serves as a second messenger in many biochemical signal-transduction pathways. However, despite the pivotal role of calcium in motile processes, it is not clear how calcium regulates overall cell movement. Here we show that transient increases in intracellular calcium, [Ca2+]i, during the locomotion of fish epithelial keratocytes, occur more frequently in cells that become temporarily 'stuck' to the substratum or when subjected to mechanical stretching. We find that calcium transients arise from the activation of stretch-activated calcium channels, which triggers an influx of extracellular calcium. In addition, the subsequent increase in [Ca2+]i is involved in detachment of the rear cell margin. Thus, we have defined a mechanism by which cells can detect and transduce mechanical forces into biochemical signals that can modulate locomotion.

Abstract. Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be ,x,2 x 10 -8 N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell-substratum contact.T o move cells must exert traction forces upon the substratum. This involves the temporal and spatial regulation of numerous force generating molecular motors. Yet an understanding of how and where moving cells generate traction forces, represents a major gap in our knowledge of cell locomotion. This paper presents the first measurements of the traction forces generated by rapidly moving cells.Traction forces produced by moving fibroblasts were first observed as distortions in flexible substrata that caused wrinkling of thin, silicone rubber films (Harris et al., 1980). These traction forces act inwards, relative to the extending lamella and retracting edge, leading to compression of the substratum such that wrinkles are formed perpendicular to the direction of lamellar extension. Wrinkles were thought to be formed by an actomyosin-based contraction of the cytoskeleton which is transmitted to the substratum via focal adhesions located just behind the extending edge and trailing cell edge. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards along the substratum. It was therefore surprising to find that traction forces generated by faster moving cell types such as leukocytes and growth cones could not be detected (Harris, 1981), since it was assumed that larger traction forces would be required for faster locomotion. However, slow moving cells such as fibroblasts form strong focal adhesions to the substratum whereas faster moving cells tend to form weaker close contacts (Couchman and Reese, 1979).In addition large numbers of actin stress fibers are found in slower moving cells, implying greater cytoskeletal contractility. Therefore rapid cell locomotion appears to rely on both weaker cell substratum adhesions and cytoskeletal contractility.To learn more about the traction forces required for rapid locomotion, we have modified the traction ...

The cytoskeletal activity of motile or adherent cells is frequently seen to induce detectable displacements of sufficiently compliant substrata. The physics of this phenomenon is discussed in terms of the classical theory of small-strain, plane-stress elasticity. The main results of such analysis is a transform expressing the displacement field of the elastic substrate as an integral over the traction field. The existence of this transform is used to derive a Bayesian method for converting noisy measurements of substratum displacement into "images" of the actual traction forces exerted by adherent or locomoting cells. Finally, the Monte Carlo validation of the statistical method is discussed, some new rheological studies of films are presented, and a practical application is given.

Moving cells display a variety of shapes and modes of locomotion, but it is not clear how motility at the molecular level relates to the locomotion of a whole cell, a problem compounded in studies of cells with complex shapes. A striking feature of fish epidermal keratocyte locomotion is its apparent simplicity. Here we present a kinematic description of locomotion which is consistent with the semicircular shape and persistent 'gliding' motion of fish epidermal keratocytes. We propose that extension of the front and retraction of the rear of these cells occurs perpendicularly to the cell edge, and that a graded distribution of extension and retraction rates along the cell margin maintains cell shape and size during locomotion. Evidence for this description is provided by the predicted circumferential motion of lamellar features and the curvature of 'photo-marked' lines within specific molecular components of moving keratocytes. Our description relates the dynamics of molecular assemblies to the movement of a whole cell.

Nanovid microscopy, which uses 30-to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 108 cm2/sec, and paucivalent probes had an average D of 0.73 x 10-8 cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 X 10-8 cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 X 10-8 cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results, Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.Nanometer-size colloidal gold probes combined with videoenhanced microscopy (nanovid microscopy) is a useful tool for studying the movements of proteins within the plasma membrane of living cells (1-7). For example, the value of nanovid microscopy already has been demonstrated for examining putative flow and transport in locomoting cells (2-7). In addition, the possibility of following the movements of individual membrane molecules has unique potential for studying the existence and size of domains in the plasma membrane (3,4,8). An important question for these new probes of motion is whether the attachment of the colloidal gold particle alters the diffusion characteristics of the bound membrane molecule. When compared with values obtained by fluorescence recovery after photobleaching (FRAP), the lateral diffusion coefficient is lower for gold-Con A on macrophages (4) but not for gold-anti-2A1-A on growth cones (5). In some instances, steric hindrance to the movements of the gold molecule could be produced by the glycocalyx, which can be as much as 50-nm thick on some cell types (9).Additionally, motion may be affected by the number of antigen binding sites on the gold probe and its degree of aggregation.In this study we characterize the effect of the colloidal gold probe on Brownian motion of memb...

Three flagellar genes of Salmonella typhimurium (flaAiI.2,flaQ, andflaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) The bacterial flagellum is a reversible rotary apparatus driven by proton motive force. Counterclockwise (CCW) rotation causes smooth swimming of the bacterium, whereas an abrupt switch to clockwise (CW) rotation causes tumbling. Bacteria migrate toward beneficial environments by modulating the frequency of switching between CCW and CW senses (see references 9 and 16 for reviews).In Salmonella typhimurium, nearly 50 genes are involved in the formation and function of the flagella (12, 14, 24). They have been placed into three categories: those necessary for flagellar formation (fla), for flagellar rotation (mot), and for modulation of switching (che). The corresponding mutant phenotypes are nonflagellate, paralyzed, and nonchemotactic, respectively.Two of the fla genes, flaAII.2 and flaQ, are particularly interesting because they give rise to several types of mutants, not only nonflagellate (the null phenotype), but also paralyzed (flaAII.2) and nonchemotactic (bothflaAII.2 and flaQ) (5,7,11,12,21,25 The flagellotropic bacteriophage X (15) was used to select

Abstract. The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coeIficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 × 10 -~° cm2/s ~< D ~< 4 × 10 -I° cm2/s) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 + 19% at 25°C (82 + 14% at 37°C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 __+ 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 + 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum.URING early embryonic development, certain groups of cells can transiently express locomotory properties that allow them to migrate long distances from their sites of origin and populate other areas of the embryo (for reviews, see references 16,41,56,57). There is a large body of evidence that suggests that cell motility results from the conjunction of environmental influences and intrinsic properties of cells. For example, the extracellular matrix glycoprotein fibronectin is known to promote cell locomotion in vitro (3,46,47,61...

Membrane protein lateral diffusion can be constrained in several ways: Diffusion can be slower than that predicted for a simple, fluid lipid bilayer; diffusion can be confined to certain regions within the total membrane; and diffusion may not be equally probable in all directions, i.e. it may be anisotropic. We know that protein diffusion is reduced by increasing concentrations of membrane proteins and by interactions of the diffusant with structure(s) peripheral to the membrane. The molecular nature of such peripheral constraints has been difficult to pinpoint, but attention is now being directed to the extracellular matrix in addition to the membrane-associated cytoskeleton. There are many proteins that are confined to lateral domains in differentiated, isolated cells and in cells organized into tissue. The mechanisms that maintain such inhomogeneous distributions should be elucidated in the next few years. Whether lateral diffusion of membrane proteins over distances of a few micrometers is usually isotropic or anisotropic will be ascertained in the near future using imaging methods combined with photobleaching.