Regulation of water flow by actin-binding protein-induced actin gelatin.

Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation.     

Receptor-mediated actin assembly in electropermeabilized neutrophils: role of intracellular pH

Neutrophil activation by a variety of stimuli is accompanied by an intracellular acidification, which has been postulated to mediate actin polymerization (Yuli and Oplatka, Science 1987, 235, 340). This hypothesis was tested using 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin staining and flow cytometry, or right angle light scattering to study actin assembly in intact and electrically permeabilized human neutrophils. Intracellular pH was measured fluorimetrically using a pH sensitive dye. In cells stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) at 21 degrees C, actin assembly clearly preceded the intracellular acidification in response to fMLP. Moreover, actin polymerization persisted in cells where intracellular pH was clamped near the resting (unstimulated) level using nigericin/K+. Finally, fMLP induced a significant increase in F-actin content in electropermeabilized neutrophils equilibrated with an extracellular medium containing up to 50 mM HEPES. These observations indicate that fMLP-stimulated F-actin assembly is not mediated by a decrease in intracellular pH and suggest that changes in transmembrane potential and ionic gradients are unlikely to mediate actin polymerization.     

Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating Swiss 3T3 fibroblasts

The formation of lamellipodia in migrating cells involves dynamic processes that occur in a cyclic manner as the leading edge of a cell slowly advances. We used video-enhanced contrast microscopy (VEC) to monitor the motile behavior of cells to classify protrusions into the temporal stages of initial and established protrusions (Fisher et al.: Cell Motility and the Cytoskeleton 11:235-247, 1988), and to monitor the fixation of cells. Multiple parameter fluorescence imaging methods (DeBiasio et al.: Journal of Cell Biology 105:1613-1622, 1987; Waggoner et al.: Methods in Cell Biology, Vol. 30, Part B, pp. 449-478, 1989) were then used to determine and to map accurately the distributions of actin, myosin and microtubules in specific types of protrusions. Initial protrusions exhibited no substructure as evidenced by VEC and actin was diffusely arranged, while myosin and microtubules were absent. Newly established protrusions contained diffuse actin as well as actin in microspikes. There was a delay in the appearance of myosin into established protrusions relative to the presence of actin. Microtubules were found in established protrusions after myosin was detected, and they were oriented parallel to the direction of migration. Actin and myosin were also localized in fibers transverse to the direction of migration at the base of initial and established protrusions. Image analysis was used to quantify the orientation of actin fibers relative to the leading edge of motile cells. The combined use of VEC, multiple parameter immunofluorescence, and image analysis should have a major impact on defining complex relationships within cells.     

Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.     

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2 ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2-carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.     

Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly.

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell-cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.     

Heparin increases mRNA levels of thrombospondin but not fibronectin in human vascular smooth muscle cells

The effects of heparin (180 micrograms/ml) on steady state mRNA levels for fibronectin, thrombospondin, actin and collagen types I and III were investigated in human umbilical artery smooth muscle cells. Heparin caused a 120% increase in thrombospondin mRNA levels and a 60% and 180% increase in the mRNA levels of procollagen chains alpha 2(I) and alpha 1(III), respectively. No change in fibronectin or actin mRNA levels resulted from heparin treatment. We reported earlier (Biochem. Biophys. Res. Comm. 148:1264, 1987) that heparin increases smooth muscle cell synthesis of both fibronectin and thrombospondin. These data show that heparin coordinately regulates thrombospondin mRNA and protein levels. The heparin induced increase in fibronectin biosynthesis apparently reflects control at the translational or post-translational level.     

Characean actin bundles as a tool for stydying actomyosin-based motility

At the inner surface of the stagnant chloroplasts of Characeae cells, bundles of actin filaments having uniform polarity are anchored. These bundles are responsible for generating the motive force of cytoplasmic streaming. It is now possible to induce movement of either beads coated with foreign myosin or organelles associated with myosin along the characean actin bundles. The Ca²⁺ sensitivities of the reconstitued movements are consistent with those of the actin-activated myosin ATPases. The use of reconstituted systems is finding wide application in the detection of various myosins in materials from which myosin is not significantly purified. Furthermore, sliding velocities and the Ca²⁺ regulation of myosins bound to organelles are now being determined. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Co-localization of components of the protein-synthesizing machinery with the cytoskeleton in G0-arrested cells.

The distribution of eukaryotic elongation factor 2 (eEF-2) in G₀-arrested fixed human skin diploid fibroblasts was studied by indirect immunofluorescent microscopy. It was found earlier that the main part of eEF-2 in cycling cells was located near the nucleus in the endoplasm (Gavrilova et al., 1987). It has been demonstrated here that the transition from proliferation to the G₀ phase of the cell cycle leads to the distribution of eEF-2 mainly along the intermediate filaments and/or microtubules. Both in cycling and in G₀-arrested fibroblasts a portion of eEF-2 is also co-localized with actin microfilament bundles. The reversion of the cells from the G₀ phase to proliferation is accompanied by rearrangement of the actin cytoskeleton and reversal to the original pattern of eEF-2 distribution. It is likely that the different types of cytoskeleton in eukaryotic cells can be involved in organization of protein-synthesizing machinery.     

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin

Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues.     

Characteristic structures of actin gels induced with hepatic actinogelin or with chicken gizzard alpha-actinin: implication for their function

We studied the properties of actinogelin, a Ca2+-regulated actin cross-linking protein isolated from Ehrlich tumor cells or rat liver. Chicken gizzard alpha-actinin was used as a Ca2+-insensitive control. Actinogelin, which has very high gelation activity under low Ca2+ conditions, was found using electron microscopic or fluorescence studies to induce formation of a characteristic structure in which actin filaments and bundles radiate to (or converge from) all directions from spot-like core structures. A similar structure was induced with actinogelin, even in the presence of 0.7 saturation of tropomyosin. No such structure was detected with actinogelin under high Ca2+ conditions, and only a few were found with gizzard alpha-actinin. Because reconstituted structures are similar to those observed intracellularly, actinogelin may be important in the formation of similar microfilament organization in the cells. It seems also important that these structures are reconstituted with only two purified protein components, i.e., actinogelin and actin. Immunocompetition studies showed that actinogelin and gizzard alpha-actinin partially shared antigenicity, and their molecular shape and peptide maps were similar. Their amino acid compositions [Kuo et al., 1982], subunit and domain structures, and binding sites on actin [Mimura and Asano, 1987] are also very similar. Therefore, it is concluded that actinogelin belongs to alpha-actinin superfamily proteins. Furthermore, the presence of functionally different subfamilies concerned with Ca2+ sensitivity, gelation-efficiency, and others is discussed. Actinogelin, which induces networks of actin filaments, may be classified as high gelation type.     

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Ga?azkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.     

Mapping the binding domain of a myosin II binding protein

The way in which actin and myosin II become localized to the contractile ring of dividing cells resulting in cleavage furrow formation and cytokinesis is unknown. While much is known about actin binding proteins and actin localization, little is known about myosin localization. A 53-kDa (53K) polypeptide present in the sea urchin egg binds to myosin II in a nucleotide-dependent manner and mediates its solubility in vitro [Yabkowitz, R., & Burgess, D.R. (1987) J. Cell Biol. 105, 927-936]. The binding site of 53K on the myosin molecule was examined in an effort to understand the mechanism of 53K-induced myosin solubility and its potential function in myosin regulation. Blot overlay and chemical cross-linking techniques utilizing myosin proteolytic fragments indicate that 53K binds to fragments proximal to the head-rod junction of myosin. Fragments distal to the head-rod junction do not bind 53K. In addition, the binding of 53K to myosin largely inhibits protease digestion that produces the head and rod fragments. The binding of 53K to the head-rod domain of myosin may be critical in regulation of myosin conformation, localization, assembly, and ATPase activity.     

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.     

Tetrahymena actin: copolymerization with skeletal muscle actin and interactions with muscle actin-binding proteins

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.     

Indirect immunofluorescence localization of ponticulin in motile cells

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.     

Structure and function of the cytoskeleton of a Dictyostelium myosin- defective mutant

To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.