Common and Specific Functions of Nonmuscle Myosin II Paralogs in Cells.

Various forms of cell motility critically depend on pushing, pulling, and resistance forces generated by the actin cytoskeleton. Whereas pushing forces largely depend on actin polymerization, pulling forces responsible for cell contractility and resistance forces maintaining the cell shape require interaction of actin filaments with the multivalent molecular motor myosin II. In contrast to muscle-specific myosin II paralogs, nonmuscle myosin II (NMII) functions in virtually all mammalian cells, where it executes numerous mechanical tasks. NMII is expressed in mammalian cells as a tissue-specific combination of three paralogs, NMIIA, NMIIB, and NMIIC. Despite overall similarity, these paralogs differ in their molecular properties, which allow them to play both unique and common roles. Importantly, the three paralogs can also cooperate with each other by mixing and matching their unique capabilities. Through specialization and cooperation, NMII paralogs together execute a great variety of tasks in many different cell types. Here, we focus on mammalian NMII paralogs and review novel aspects of their kinetics, regulation, and functions in cells from the perspective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cells.

Dendritic organization of actin comet tails.

Polymerization of actin filaments is necessary for both protrusion of the leading edge of crawling cells and propulsion of certain intracellular pathogens, and it is sufficient for generating force for bacterial motility in vitro. Motile intracellular pathogens are associated with actin-rich comet tails containing many of the same molecular components present in lamellipodia, and this suggests that these two systems use a similar mechanism for motility. However, available structural evidence suggests that the organization of comet tails differs from that of lamellipodia. Actin filaments in lamellipodia form branched arrays, which are thought to arise by dendritic nucleation mediated by the Arp2/3 complex. In contrast, comet tails have been variously described as consisting of short, randomly oriented filaments, with a higher degree of alignment at the periphery, or as containing long, straight axial filaments with a small number of oblique filaments. Because the assembly of pathogen-associated comet tails has been used as a model system for lamellipodial protrusion, it is important to resolve this apparent discrepancy. Here, using a platinum replica approach, we show that actin filament arrays in comet tails in fact have a dendritic organization with the Arp2/3 complex localizing to Y-junctions as in lamellipodia. Thus, comet tails and lamellipodia appear to share a common dendritic nucleation mechanism for protrusive motility. However, comet tails differ from lamellipodia in that their actin filaments are usually twisted and appear to be under significant torsional stress.

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Actin machinery: pushing the envelope.

The reconstitution of microbial rocketing motility in vitro with purified proteins has recently established definitively that no myosin motor is required for protrusion. Instead, actin polymerization, in conjunction with a small number of proteins, is sufficient. A dendritic pattern of nucleation controlled by the Arp2/3 complex provides an efficient pushing force for lamellipodial motility.

Two components of actin-based retrograde flow in sea urchin coelomocytes.

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Speckle microscopic evaluation of microtubule transport in growing nerve processes.

Assembly of microtubules is fundamental to neuronal morphogenesis. Microtubules typically form crosslinked bundles in nerve processes, precluding resolution of single microtubules at the light microscopic level. Therefore, previous studies of microtubule transport in neurites have had to rely on indirect approaches. Here we show that individual microtubules can be visualized directly in the axonal shafts of Xenopus embryo neurons by using digital fluorescence microscopy. We find that, although the array of axonal microtubules is dynamic, microtubules are stationary relative to the substrate. These results argue against a model in which newly synthesized tubulin is transported down the axon in the form of microtubules.

Progress in protrusion: the tell-tale scar.

The crawling movement of a cell involves protrusion of its leading edge, in coordination with the translocation of its cell body, and depends upon a cytoplasmic machinery able to respond to signals from the environment. Protrusion is now understood to be driven by actin polymerization, and signalling from membrane receptors to actin has been shown to be mediated by the Rho family of GTPases. However, a major gap in our understanding of regulated motility has been how to connect the signalling pathway to the motile machinery itself. Recent structural, biochemical and genetic studies have identified some of the missing links and provided a strong working model for the pathways and mechanisms by which the signals are interpreted and implemented.

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

Network contraction model for cell translocation and retrograde flow.

Kinetic and structural analysis of the actin-myosin II system in mammalian fibroblasts and fish epidermal keratocytes suggests that the cell's motility machinery arises behind the leading edge in the form of myosin filament clusters immersed in an actin filament network. We discuss how the contraction of this actin-myosin II network is related to the formation of actin-myosin filament bundles, cell translocation and retrograde flow.

Self-polarization and directional motility of cytoplasm.

BACKGROUND: Directional cell motility implies the presence of a steering mechanism and a functional asymmetry between the front and rear of the cell. How this functional asymmetry arises and is maintained during cell locomotion is, however, unclear. Lamellar fragments of fish epidermal keratocytes, which lack nuclei, microtubules and most organelles, present a simplified, perhaps minimal, system for analyzing this problem because they consist of little other than the motile machinery enclosed by a membrane and yet can move with remarkable speed and persistence. RESULTS: We have produced two types of cellular fragments: discoid stationary fragments and polarized fragments undergoing locomotion. The organization and dynamics of the actin-myosin II system were isotropic in stationary fragments and anisotropic in the moving fragments. To investigate whether the creation of asymmetry could result in locomotion, a transient mechanical stimulus was applied to stationary fragments. The stimulus induced localized contraction and the formation of an actin-myosin II bundle at one edge of the fragment. Remarkably, stimulated fragments started to undergo locomotion and the locomotion and associated anisotropic organization of the actin-myosin II system were sustained after withdrawal of the stimulus. CONCLUSIONS: We propose a model in which lamellar cytoplasm is considered a dynamically bistable system capable of existing in a non-polarized or polarized state and interconvertible by mechanical stimulus. The model explains how the anisotropic organization of the lamellum is maintained in the process of locomotion. Polarized locomotion is sustained through a positive-feedback loop intrinsic to the actin-myosin II machinery: anisotropic organization of the machinery drives translocation, which then reinforces the asymmetry of the machinery, favoring further translocation.

Functional coordination of microtubule-based and actin-based motility in melanophores.

The fish melanophore has been considered the exemplar of microtubule-based organelle transport. In this system, a radial array of uniformly polarized microtubules [1] provides a framework on which dynein-related and kinesin-related motors drive pigment granules toward the minus or plus ends, respectively [2-4]. Stimulation of minus-end motors accounts satisfactorily for aggregation of granules at the cell center. Rapid dispersion is clearly microtubule-dependent; however, the uniform distribution of granules throughout the cytoplasm is paradoxical because stimulation of plus-end motors is predicted to drive the granules to the cell margin. This paradox suggested that the transport system was incompletely understood. Here, we report the discovery of a microtubule-independent motility system in fish melanophores. The system is based on actin filaments and is required for achieving uniform distribution of pigment granules. When it is abrogated, granules accumulate at the cell's margin as predicted for microtubule plus-end motors acting alone. The results presented here demonstrate the functional coordination of microtubule and actin filament systems, a finding that may be of general significance for organelle motility in cytoplasm.

Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation.

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin-myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin-myosin network in the lamellipodial/cell body transition zone.

Polarity sorting of actin filaments in cytochalasin-treated fibroblasts.

The polarity of actin filaments is fundamental for the subcellular mechanics of actin-myosin interaction; however, little is known about how actin filaments are oriented with respect to myosin in non-muscle cells and how actin polarity organization is established and maintained. Here we approach these questions by investigating changes in the organization and polarity of actin relative to myosin II during actin filament translocation. Actin and myosin II reorganization was followed both kinetically, using microinjected fluorescent analogs of actin and myosin, and ultrastructurally, using myosin S1 decoration and immunogold labelling, in cultured fibroblasts that were induced to contract by treatment with cytochalasin D. We observed rapid (within 15 minutes) formation of ordered actin filament arrays: short tapered bundles and aster-like assemblies, in which filaments had uniform polarity with their barbed ends oriented toward the aggregate of myosin II at the base of a bundle or in the center of an aster. The resulting asters further interacted with each other and aggregated into bigger asters. The arrangement of actin in asters was in sharp contrast to the mixed polarity of actin filaments relative to myosin in non-treated cells. At the edge of the cell, actin filaments became oriented with their barbed ends toward the cell center; that is, the orientation was opposite to what was observed at the edge of nontreated cells. This rearrangement is indicative of relative translocation of actin and myosin II and of the ability of myosin II to sort actin filaments with respect to their polarity during translocation. The results suggest that the myosin II-actin system of non-muscle cells is organized as a dynamic network where actin filament arrangement is defined in the course of its interaction with myosin II.

Cytoplasmic assembly of microtubules in cultured cells.

The origin of non-centrosomal microtubules was investigated in a variety of animal cells in culture by means of time-lapse digital fluorescence microscopy. A previous study (Keating et al. (1997) Proc. Nat. Acad. Sci. USA 94, 5078-5083) demonstrated a pathway for formation of non-centrosomal microtubules by release from the centrosome. Here we show a parallel pathway not dependent upon the centrosome. Correlative immunostaining with anti-tubulin antibodies and electron microscopy established that apparent free microtubules observed in vivo were not growing ends of long stable microtubules. Free microtubules appeared spontaneously in the cytoplasm and occasionally by breakage of long microtubules. Estimates of the frequencies of free microtubule formation suggest that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of long microtubules generated nascent plus and minus ends; the nascent minus end was generally stable while the plus end was always dynamic. The stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. Parameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indicating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave apparent diffusion coefficients for free and long microtubules which permitted an estimate of turnover half-times. The results support the concept that, in PtK1 cells, a pathway other than plus end dynamics is needed to account for the rapidity of microtubule turnover.

Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton.

By immunogold labeling, we demonstrate that "millipede-like" structures seen previously in mammalian cell cytoskeletons after removal of actin by treatment with gelsolin are composed of the cores of vimentin IFs with sidearms containing plectin. These plectin sidearms connect IFs to microtubules, the actin-based cytoskeleton and possibly membrane components. Plectin binding to microtubules was significantly increased in cells from transgenic mice lacking IFs and was reversed by microinjection of exogenous vimentin. These results suggest the existence of a pool of plectin which preferentially associates with IFs but may also be competed for by microtubules. The association of IFs with microtubules did not show a preference for Glu-tubulin. Nor did it depend upon the presence of MAP4 since plectin links were retained after specific immunodepletion of MAP4. The association of IFs with stress fibers survived actin depletion by gelsolin suggesting that myosin II minifilaments or components closely associated with them may play a role as plectin targets. Our results provide direct structural evidence for the hypothesis that plectin cross-links elements of the cytoskeleton thus leading to integration of the cytoplasm.

Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells.

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.

Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles.

The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion.

Transverse pattern of microfilament bundles induced in epitheliocytes by cylindrical substrata.

Cylindrical culture substrata are known to induced longitudinal orientation of polarized fibroblasts and corresponding alignment of actin microfilament bundles in these cells. We studied microfilament bundle distribution in two cell types, fibroblasts and epitheliocytes, spread on two kinds of anisotropic substrata, quartz glass cylinders with a diameter 32 microns and narrow (25-40 microns wide) flat glass adhesive strips with non-adhesive borders. Rat embryo and human diploid fibroblasts, as expected, formed predominantly longitudinally aligned bundles on both substrata. In contrast, transverse bundles on cylinders and randomly oriented bundles on flat strips were formed in IAR-2 and MDCK epithelial cells. We interpret these data as showing that the epitheliocyte attempts to override the guiding influence of anisotropic substrata. The microfilament bundle pattern on cylinders depends on the integrity of the microtubules. Colcemid-induced microtubule depolymerization caused formation of longitudinal as well as transverse bundles both in fibroblasts and epitheliocytes, thus diminishing the differences in microfilament bundle patterns in two cell types. These results show that microtubules control the cell-type-specific distribution of microfilament bundles both in polarized fibroblasts and in discoid epitheliocytes. However, the results of this control are opposite: microtubules enhance cell polarization in fibroblasts, but prevent it in epithelial cells.