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 ...

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.

Fish keratocytes can generate rearward directed traction forces within front portions of the lamellipodium, suggesting that a retrograde flow of actin may also occur here but this was not detected by previous photoactivation experiments. To investigate the relationship between retrograde flow and traction force generation, we have transfected keratocytes with GFP-actin and used fluorescent speckle microscopy, to observe speckle flow. We detected a retrograde flow of actin within the leading lamellipodium that is inversely proportional to both protrusion rate and cell speed. To observe the effect of reducing contractility, we treated transfected cells with ML7, a potent inhibitor of myosin II. Surprisingly, ML7 treatment led to an increase in retrograde flow rate, together with a decrease in protrusion and cell speed, but only in rapidly moving cells. In slower moving cells, retrograde flow decreased, whereas protrusion rate and cell speed increased. These results suggest that there are two mechanisms for producing retrograde flow. One involves slippage between the cytoskeleton and adhesions, that decreases traction force production. The other involves slippage between adhesions and the substratum, which increases traction force production. We conclude that a biphasic relationship exists between retrograde actin flow and adhesiveness in moving keratocytes.

The budding of the urogenital sinus epithelium into the surrounding mesenchyme signals the onset of prostate morphogenesis. The epithelial and mesenchymal factors that regulate ductal budding and the ensuing process of ductal growth and branching are not fully known. We provide evidence that bone morphogenetic protein 4 (BMP4) is a mesenchymal factor that regulates ductal morphogenesis. The Bmp4 gene was most highly expressed in the male urogenital sinus from embryonic day 14 through birth, a period marked by formation of main prostatic ducts and initiation of ductal branching. From an initial wide distribution throughout the prostatic anlage of the urogenital sinus, Bmp4 expression became progressively restricted to the mesenchyme immediately surrounding the nascent prostatic ducts and branches. Exogenous BMP4 inhibited epithelial cell proliferation and exhibited a dose-dependent inhibition of ductal budding in urogenital sinus tissues cultured in vitro. Adult Bmp4 haploinsufficient mice exhibited an increased number of duct tips in both the ventral prostate and coagulating gland. Taken together, our data indicate that BMP4 is a urogenital sinus mesenchymal factor that restricts prostate ductal budding and branching morphogenesis.