Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.

Two-step positioning of a cleavage furrow by cortexillin and myosin II.

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.

Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position.

Cytoplasmic dynein is a microtubule-based motor protein responsible for vesicle movement and spindle orientation in eukaryotic cells. We show here that dynein also supports microtubule architecture and determines centrosome position in interphase cells. Overexpression of the motor domain in Dictyostelium leads to a collapse of the interphase microtubule array, forming loose bundles that often enwrap the nucleus. Using green fluorescent protein (GFP)-alpha-tubulin to visualize microtubules in live cells, we show that the collapsed arrays remain associated with centrosomes and are highly motile, often circulating along the inner surface of the cell cortex. This is strikingly different from wild-type cells where centrosome movement is constrained by a balance of tension on the microtubule array. Centrosome motility involves force-generating microtubule interactions at the cortex, with the rate and direction consistent with a dynein-mediated mechanism. Mapping the overexpression effect to a C-terminal region of the heavy chain highlights a functional domain within the massive sequence important for regulating motor activity.

Patterns of cellular activities based on protein sorting in cell motility, endocytosis and cytokinesis.

In order to move persistently, a cell has to harmonize its protrusion and retraction with attachment and detachment from the substrate. Time-series analyses based on fluctuations in these activities are being used in combination with advanced imaging techniques to unravel the network of protein-protein interactions that tune the activities in a motile cell and co-ordinate them in space and time. Fusions with the green fluorescent protein have helped to visualize the recruitment of cytoskeletal proteins from a soluble pool and their transient assembly into supramolecular structures. Using a series of mutants deficient in specific cytoskeletal proteins has revealed common themes and interrelationships between cell motility, endocytosis and cytokinesis. For instance, a phagocytic cup competes with leading-edge formation, and recruits the same actin-associated proteins. Cytokinesis is based on the fine tuning of activities in the microtubule system and the actin network in the cell cortex. Cells dividing on a substrate apply tension to the surface on which they adhere, as determined by the silicone rubber technique. Actin-associated proteins are sorted during cytokinesis either to the extensions formed at the poles of a dividing cell or to the cleavage furrow. A major effort will be required to elucidate the mechanisms that dictate the pattern of local activities and drive the translocation of proteins in cell motility, endocytosis and cytokinesis.

Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells.

To study centrosome motility and the interaction of microtubules with the cell cortex in mitotic, post-mitotic and interphase cells, (alpha)-tubulin was tagged in Dictyostelium discoideum with green fluorescent protein. Multinucleate cells formed by myosin II-null mutants proved to be especially suited for the analysis of the control of cleavage furrow formation by the microtubule system. After docking of the mitotic apparatus onto the cell cortex during anaphase, the cell surface is activated to form ruffles on top of the asters of microtubules that emanate from the centrosomes. Cleavage furrows are initiated at spaces between the asters independently of the positions of spindles. Once initiated, the furrows expand as deep folds without a continued connection to the microtubule system. Occurrence of unilateral furrows indicates that a closed contractile ring is dispensable for cytokinesis in Dictyostelium. The progression of cytokinesis in the multinucleate cells underlines the importance of proteins other than myosin II in specifying a cleavage furrow. The analysis of centrosome motility suggests a major role for a minus-end directed motor protein, probably cytoplasmic dynein, in applying traction forces on guiding microtubules that connect the centrosome with the cell cortex.

Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system and formation of the cleavage furrow.

Mitosis was studied in multinucleated and mononucleated mutant cells of Dictyostelium discoideum that lack myosin II (Manstein et al. (1989) EMBO J. 8, 923-932). Multinucleated cells were produced by culture in suspension, mononucleated cells were enriched by growth on a solid surface (DeLozanne and Spudich (1987) Science 236, 1086-1091). The multinucleated cells disclosed interactions of mitotic complexes with the cell cortex that were not apparent in normal, mononucleated cells. During the anaphase stage, entire mitotic complexes consisting of spindle, microtubule asters, and separated sets of chromosomes were translocated to the periphery of the cells. These complexes were appended at a distance of about 3 microns from the cell surface, in a way that the spindle became orientated in parallel to the surface. Subsequently, lobes of the cell surface were formed around the asters of microtubules. These lobes were covered with tapered protrusions rich in coronin, an actin associated protein that typically accumulates in dynamic cell-surface projections (DeHostos et al. (1991) EMBO J. 10, 4097-4104). During their growth on a solid surface, mononucleated myosin II-null cells passed through the mitotic cleavage stages with a speed comparable to wild-type cells. Cytokinesis as linked to mitosis is distinguishable by several parameters from traction mediated cytofission, which results in the pinching off of pieces of a multinucleated cell in the interphase. The possibility is discussed that cells can cleave during mitosis without forming a contractile ring at the site of the cleavage furrow.

Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells.

Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123-137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086-1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081-1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328-331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein-actin fusion.