Abstract. The actin-based cytoskeleton is a dynamic component of living cells with major structural and contractile properties involved in fundamental cellular processes . The action of actin-binding proteins can decrease or increase the gel structure . Changes in the actin-based cytoskeleton have long been thought to modulate the myosin II-based contractions involved in these cellular processes, but there has been some debate concerning whether maximal gelation increases or decreases contractile activity. To address this question, we have examined how contractile activity is modulated by the extent of actin gelation . The model system consists of physiologically relevant concentrations and molar ratios of actin filaments (whose lengths are controlled by gelsolin), the actin-cross-linking protein filamin, and smooth muscle myosin II. This system has been F OR over 150 years biologists have studied the correlation between gel-sol transitions of the cytomatrix and cell movement . Recognition of the structure of cytoplasm, changes in the consistency ofthis structure, and the relationship between these changes and contractility in whole cells led to initial theories ofamoeboid movement (for reviews see Allen, 1973;and Taylor and Condeelis, 1979) . The study of nonmuscle cell extracts extended the understanding ofthe relationship between gelation and contraction and stimulated the identification ofcritical proteins for these processes (Taylor and Condeelis, 1979;Stossel, 1982; Stossel et al., 1985, Pollard andCooper, 1986) . The identification of the proteins involved in gelation, solation, and contraction enabled the reconstitution of these processes in vitro . Reconstitutions allowed selected and controlled conditions to be defined in order to explore the molecular basis of the phenomenology. Two major hypotheses emerged from the early experiments exploring the relationship between the state of gelation of the actin-based cytoplasmic gel and contractility. Taylor and colleagues (see Taylor and Fechheimer, 1982) proposed that a decrease in the gel structure (solation) decreased the resistance of sliding actin filaments, thus increasing the rate of contraction . The solation could result from a decrease in actin filament lengths and/or a decrease in the ® The Rockefeller University Press, 0021-9525/91/09/1005/11 $2 .00 TheJournal of Cell Biology, Volume 114, Number 5, September 19911005-1015 1005 studied at the macroscopic and light microscopic levels to relate the gel structure to the rate of contraction . We present results which show that while a minimal amount of structure is necessary to transmit the contractile force, increasing the gel structure inhibits the rate of contraction, despite an increase in the actinactivated Mgz+ATPase activity of myosin . Decreasing the total myosin concentration also inhibits the rate of contraction . Application of cytochalasin D to one side of the contractile network increases the rate of contraction and also induces movement comparable to flare streaming observed in isolated a...

Abstract. Serum-deprived Swiss 3T3 fibroblasts constitutively form stress fibers at their edges. These fibers move centripetally towards the perinuclear region where they disassemble. Serum stimulation causes shortening of fibers in a manner suggesting active actin-myosin-based contraction (Giuliano, K. A. and D. L. Taylor. 1990. Cell Motil. and Cytoskeleton . 16:14-21). To elucidate the role of actin-based gel structure in these movements, we examined the effects of disrupting actin organization with cytochalasin . Serum-deprived fibroblasts were microinjected with rhodamine analogs of actin or myosin II and fiber dynamics were monitored with a multimode light microscope workstation using video-enhanced contrast and fluorescence modes. When cells were perfused with >3 I,M cytochalasin B or 0.5 uM cytochalasin D, formation and transport of stress fibers were reversibly inhibited, and rapid and immediate shortening of existing fibers was induced. Quantification of actin and N a simplified model of cell crawling suggested by the behavior of giant amoebae, locomotion is believed to be brought about by the following cycle of events : solated cytoplasm (endoplasm) streams forward to the leading edge, where it is recruited into a cortical gel (ectoplasm). The cortical gel exhibits a rearward transport away from the leading edge, with the leading edge being determined, in part, by a partial decrease in gelation . The rearward transport involves a contraction and results in the exertion of a tractional force on the substratum via linkages across the plasma membrane . Contraction is coupled to solation of the cortical gel, with the solated components being recruited into the forward stream and driven forward under positive hydrostatic pressure to continue the cycle (Taylor and Condeelis, 1979;Taylor and Fechheimer, 1982).An essential element of this model is the rearward movement of material at or near the cell surface. Such movement has been dubbed "ectoplasmic contractionby those studying amoebae (for reviews see Taylor and Condeelis, 1979;and Taylor and Fechheimer, 1982) and "cortical flow" by others (Bray and White, 1988 myosin II fluorescence associated with individual shortening fibers demonstrated that fluorescence per length of fiber increased for both components, suggesting sliding filament contraction . However, there was also a net loss of both actin and myosin II from fibers as they shortened, indicating a self-destructive process. Loss of material from fibers coupled with increased overlap of actin and myosin II remaining in the fibers suggested that contraction could be induced not only by increasing the force exerted by contractile motors, but also by decreasing gel structure through partial solation. Finally, cytochalasin accelerated contraction of actin-myosin-based gels reconstituted from purified proteins in the absence of myosin-based regulation, further supporting solation-contraction coupling as a possible mechanism for modulating cytoplasmic contractility (Taylor, D. L . and M. Fechheimer. 1982 ....

Abstract. We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contrac-

Regulation of actin/myosin II force generation by calcium [Kamm and Stull, Annu. Rev. Physiol. 51:299-313, 1989] and phosphorylation of myosin II light chains [Sellers and Adelstein, "The Enzymes," Vol. 18, Orlando, FL: Academic Pres, 1987, pp. 381-418] is well established. However, additional regulation of actin/myosin II force generation/contraction may result from actin-binding proteins [Stossel et al., Ann. Rev. Cell Biol. 1:353-402, 1985; Pollard and Cooper, Ann. Rev. Biochem. 55:987-1035, 1986] as they affect the gel state of the actin cytomatrix [reviewed in Taylor and Condeelis, Int. Rev. Cytol., 56:57-143, 1979]. Regulation of the gel state of actin may determine whether an isotonic or isometric contraction results from the interaction between myosin and actin. We have extended the single actin filament motility assay of Kron and Spudich [Proc. Natl. Acad. Sci. U.S.A. 83:6272-6276, 1986] by including filamin or alpha-actinin on the substrate with myosin II to examine how actin-crosslinking proteins regulate the movements of single actin filaments. Increasing amounts of actin-crosslinking proteins inhibit filament velocity and decrease the number of filaments moving. Reversal of crosslinking yields increased velocities and numbers of moving filaments. These results support the solation-contraction coupling hypothesis [see Taylor and Fechheimer, Phil. Trans. Soc. London B 299:185-197, 1982] which proposes that increased crosslinking of actin inhibits myosin-based contraction. This study also illustrates the potentially varied roles of different actin-crosslinking proteins and offers a novel method to examine actin-binding protein activity and their regulation of motility at the single molecule level.

Subdomains of the cytoplasmic volume in tissue culture cells exclude large tracer particles relative to small. Evidence suggests that exclusion of the large particles is due to molecular sieving by the dense meshwork of microfilaments found in these compartments, but exclusion as a result of the close apposition of the dorsal and ventral plasma membrane of the cell in these regions has not been ruled out conclusively. In principle, these two mechanisms can be distinguished by the dependence of exclusion on tracer particle size. By fluorescence ratio imaging we have measured the partition coefficient (P/PO) into excluding compartments for tracer particles ranging in radius from 1 to 41 nm. The decay of P/PO as a function of particle radius is better fitted by three molecular sieving models than by a slit pore model. The sieving models predict a percolation cutoff radius of the order of 50 nm for partitioning into excluding compartments.