Actin Filament Arrays Research Articles

Leading edge: whY-actin branches attract Arp2/3?

Actin dynamics play a central role in the extension of the leading edge of crawling cells and neurons as well as in the motility of certain parasites. Forward movement is driven by actin growth from the barbed end, close to the membrane, coupled with depolymerization from the pointed end, causing a directional flow of actin monomers. Work on GTPases has shown that the organization of filamentous actin into a lamellipodial dendritic brush requires Rac, filopodial bundle formation requires Cdc42, and actin stress fibre formation requires Rho. But, what is the fine-structure of actin at the leading edge? This clearly must be important – considering the mechanical work required to push the membrane forward. The textbook shows that long actin filaments protrude at the leading edge. But how can this create a pushing force, and is this so?Following up on previous work, Svitkina and Borisy show that actin forms a very structured and finely meshed network of short filaments at the leading edge1xArp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. Svitkina, T.M. and Borisy, G.G. J. Cell Biol. 1999; 145: 1009–1026Crossref | PubMed | Scopus (699)See all

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.

Functional design in the actin cytoskeleton

Changes in cell shape, anchorage and motility are all associated with the dynamic reorganisation of the architectural arrays of actin filaments that make up the actin cytoskeleton. The relative expression of these functionally different actin filament arrays is intimitely linked to the pattern of contacts that a cell develops with its extracellular substrate. Cell polarity is acquired by the development of an asymmetric pattern of substrate contacts, effected in a specific, site-directed manner by the delivery of adhesion-site modulators along microtubules.

Microtubule and actin filament organization during stomatal morphogenesis in the fern Asplenium nidus. II. Guard cells.

The post-cytokinetic guard cells of Asplenium nidus display a prominent perinuclear microtubule system and a few microtubules under the periclinal walls. Afterwards, microtubules appear on the whole surface of the ventral wall, whereas those below the periclinal walls proliferate and tend to become parallel with the ventral wall. The perinuclear microtubules gradually diminish but persist in later stages of guard cell differentiation. In post-cytokinetic guard cells, actin is found in the perinuclear cytoplasm and in the cortical cytoplasm lining all the walls. In differentiating guard cells, the following cortical microtubules and actin filament 'systems' appear in succession: (a) radial microtubule and actin filament arrays beneath the periclinal walls converging on the stomatal pore region, (b) anticlinal microtubule bundles, which are co-localized with actin filaments, along the ventral wall outlining the region of the stomatal pore, (c) periclinal microtubules and actin filaments on the polar ventral wall ends. These cytoskeletal systems, except for the radial actin filaments, persist in advanced stages of guard cell differentiation. Instead of the radial actin filaments, a prominent actin filament reticulum is organized under the margins of the developing wall thickenings of the stomatal pore. In addition, an extensive endoplasmic actin filament reticulum develops around the plastids. It seems likely that the successive microtubule systems in guard cells are formed by putative microtubule organizing centres operating in a definite spatial and temporal succession. Guard cell morphogenesis is the outcome of a definite process, in which the cortical microtubule cytoskeleton plays the primary role, implicated in the deposition of cellulose microfibrils and probably of the local wall thickenings. Callose or a callose-like glucan is deposited on the whole surface of the nascent ventral wall and in the wall regions where thickenings are deposited. Finally, the guard cells of Asplenium assume a kidney shape and display polar hypostomatic swellings. Particular structural features established in guard cell mother cells affect guard cell morphogenesis.

How to analyze electron micrographs of rafts of actin filaments crosslinked by actin-binding proteins

Actin bundles are common cytoskeletal structures but ones which are usually polymorphic, varying from bundle to bundle. Two-dimensional arrays of actin filaments crosslinked by actin-bundling proteins are more tractable structures to analyze than are the three-dimensional bundles found in cells. The first step in analyzing these two-dimensional “rafts” is to determine the spatial relationships between neighboring filaments. It is difficult to discern such relationships by inspection of the electron micrographs of rafts, but easy by examination of the Fourier transforms. We provide theory and examples of the analysis of transforms of rafts, and show that different bundling proteins give rise to different kinds of rafts.

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P 0, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.

Interaction between G-Actin and Various Types of Liposomes: A 19F, 31P, and 2H Nuclear Magnetic Resonance Study

We have investigated in the present study the interaction between G-actin and various types of liposomes, zwitterionic, positively charged, and negatively charged. To investigate at the molecular level the conformation of actin in the presence of lipids, we have selectively attached a fluorinated probe, 3-bromo-1,1,1-trifluoropropanone, to the actin cysteine residues 10, 285, and 374 and used high-resolution 19F nuclear magnetic resonance spectroscopy to investigate the probe resonances. The results indicate a change in the mobility of the 19F labels when G-actin is in the presence of positively charged liposomes made of DMPC and stearylamine and in the presence of DMPG, a negatively charged lipid. No conformational change was observed in the actin molecule in the presence of neutral liposomes. Electron micrographs of these systems reveal the formation of paracrystalline arrays of actin filaments at the surface of the positively charged liposomes, while no evidence of actin polymerization or paracrystallization was observed in the presence of DMPG. The interaction between actin and the lipid polar headgroup has also been investigated using solid-state phosphorus and deuterium NMR. The results indicate no evidence of interaction between actin and zwitterionic liposomes but show an interaction between the positively charged liposomes and a negative charge on the actin molecules. Interestingly, the negatively charged liposomes interact with a positive charge, which is most likely associated with the three residues (His-Arg-Lys) preceding the cysteine 374 residue in the protein.

Force-velocity relation of sliding of skeletal muscle myosin, arranged on a paramyosin filament, on actin cables.

To investigate in vitro ATP-dependent sliding of regularly arranged myosin molecules on actin filaments, we prepared thick hybrid filaments in which myosin molecules isolated from rabbit skeletal muscle were arranged around the paramyosin core (length, 10-20 micron; diameter, </=0.2 micron) obtained from a molluscan smooth muscle. A single to a few thick hybrid filaments were attached to a polystyrene bead (diameter, 4.5 micron; specific gravity, 1.5) and made to slide on actin filament arrays (actin cables) in the internodal cell of an alga, mounted on the rotor of a centrifuge microscope. The bead was subjected to centrifugal forces serving as external loads to the ATP-dependent actin-myosin sliding. The maximum unloaded sliding velocity of the thick filament attached-bead (mean, 3.4 micron/s; 20-23 degrees C) was significantly higher than that of the bead coated with randomly oriented myosin molecules reported previously. The steady-state force-velocity (P-V) relations obtained were qualitatively similar to those in intact skeletal muscle fibers. These results indicate that this in vitro motility assay system retains the basic characteristics of contracting skeletal muscle fibers, and that it may be effectively used to study mechanisms underlying the steady-state P-V characteristics of ATP-dependent actin-myosin sliding using various recombinant myosins produced in nonmuscle cells.

Evidence for the essential role of myosin subfragment-2 in the ATP-dependent actin-myosin sliding in muscle contraction.

The role of myosin subfragment-2 (myosin S-2) in muscle contraction was studied by using an in vitro motility assay system in which the ATP-dependent sliding between myosin-coated polystyrene beads and actin filament arrays (actin cables) of giant algal cells were recorded under constant external loads provided with a centrifuge microscope. With antibody to myosin S-2 below 0.3 mg/ml, the maximum "isometric" force generated by myosin molecules on the bead decreased markedly, but the unloaded bead-sliding velocity along actin cables did not change appreciably, indicating a decrease in the number of myosin molecules interacting with actin cables. The antibody at 0.3-1.5 mg/ml decreased not only the maximum isometric force, but also the unloaded bead-sliding velocity in a dose-dependent manner. With the antibody at 1.5-3 mg/ml, the beads eventually stopped moving to remain attached to actin cables. These beads could be readily detached from actin cables with very small centrifugal forces, indicating very weak actin-myosin linkages. The antibody had no effect on rigor actin-myosin linkages formed before the antibody application. These results are consistent with the view that myosin S-2 plays an essential role in muscle contraction.

Cytoskeletal characterization of arteriovenous epithelioid cells

Data on the cytoskeleton of epithelioid cells in arteriovenous anastomosis (AVA) are sparse, but there is evidence that the (myo)-epithelioid cells of the AVAs represent a specialized smooth muscle cell type with less contractile properties. We demonstrated the expression of alpha-smooth muscle actin, smooth muscle myosin, calponin, caldesmon, and caveolin in epithelioid cells of rabbit ear and in human toes, finger tips, and glomus tumors by means of indirect immunofluorescence techniques and immunoelectron microscopy. Epithelioid cells in rabbit ear did not express vimentin, but it was present in human toes, finger tips, and glomus tumors. Epithelioid cells in human toes, finger tips, and glomus tumors did not express desmin, but it was present in rabbit ear. Epithelioid cells did not express cytokeratins. The epithelioid cells examined showed only a weak expression of the protein smoothelin, which occurs exclusively in contractile smooth muscle cells. Immunoelectron microscopical demonstration of (alpha-smooth muscle actin revealed a striking difference in the arrangement of actin filaments in the epithelioid cells as compared to that in the smooth muscle cells of blood vessels. The epithelioid cells contained a loose array of actin filaments, whereas the smooth muscle cells contained tightly packed parallel actin bundles. In the present study we observed a correlation between the lack of contractile marker protein expression in epithelioid cells and the presence of only a few filaments, although the epithelioid cells are alpha-smooth muscle actin positive. The reduced number of contractile elements in the epithelioid cells of rabbit and human anastomoses suggests a lower contractility of epithelioid cells compared to that of the surrounding smooth muscle cells in anastomoses. A second interesting difference between both cell types is the high number of caveolae in epithelioid cells. Immunoelectron microscopy showed a compact distribution of caveolae at the epithelioid cell border, but a more dispersed distribution of caveolae in the cytoplasm of the blood vessel endothelium. The benign glomus tumor was characterized by an expression pattern of cytoskeletal proteins similar to that of epithelioid cells, confirming its description as a benign tumor.

Changes in actin filament arrays in protocorm cells of the orchid species, Spiranthes sinensis, induced by the symbiotic fungus Ceratobasidium cornigerum

Seeds of the terrestrial orchid, Spiranthes sinensis, were germinated in vitro in association with the symbiotic fungus, Ceratobasidium cornigerum. Resulting colonized protocorms were prepared for light microscopy, transmission electron microscopy, and fluorescence labelling of actin filaments for examination with laser scanning confocal microscopy. Fungal hyphae invaded the suspensor end of embryos, formed typical hyphal coils (pelotons) within parenchyma cells, and then underwent lysis resulting in degraded hyphal masses. Hyphae and hyphal masses were enveloped by host-derived membrane. Changes in actin filament arrays accompanied fungal colonization. Uncolonized cells had a network of actin filaments and actin bundles (cables) located in the cortical region of the cell cytoplasm; some of these were associated with the nucleus and amyloplasts. Although actin filament arrays were still present in protocorm cell cytoplasm during fungal entry and peloton formation, most of the cortical network disappeared and instead actin filaments radiated from the periphery of developing pelotons towards the cell wall. Degraded hyphal masses also had actin filament arrays associated with them, again radiating toward the cell periphery; a network of cortical actin filaments reappeared in the protocorm cell cytoplasm at this stage. Actin filaments did not appear to have a close physical association with fungal hyphae except in the epidermal hairs that developed from protocorms; this differs from our previous observations on microtubules in this system. Key words: actin, actin filaments, orchids, mycorrhizas, laser scanning confocal microscopy.

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.

The Chlamydomonas mating type plus fertilization tubule, a prototypic cell fusion organelle: isolation, characterization, and in vitro adhesion to mating type minus gametes.

In the biflagellated alga Chlamydomonas, adhesion and fusion of the plasma membranes of gametes during fertilization occurs via an actin-filled, microvillus-like cell protrusion. Formation of this approximately 3-microm-long fusion organelle, the Chlamydomonas fertilization tubule, is induced in mating type plus (mt+) gametes during flagellar adhesion with mating type minus (mt-) gametes. Subsequent adhesion between the tip of the mt+ fertilization tubule and the apex of a mating structure on mt- gametes is followed rapidly by fusion of the plasma membranes and zygote formation. In this report, we describe the isolation and characterization of fertilization tubules from mt+ gametes activated for cell fusion. Fertilization tubules were detached by homogenization of activated mt+ gametes in an EGTA-containing buffer and purified by differential centrifugation followed by fractionation on sucrose and Percoll gradients. As determined by fluorescence microscopy of samples stained with a fluorescent probe for filamentous actin, the method yielded 2-3 x 10(6) fertilization tubules/microg protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical ends of activated mt- gametes between the two flagella, the site of the mt- mating structure; a single fertilization tubule bound per cell, binding was specific for gametes, and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt- gametes.

An in vitro motility assay system retaining the steady-state force-velocity characteristics of muscle fibers under positive and negative loads

To investigate in vitro myofilament sliding in conditions close to those in muscle fibers, we developed a new assay system in which a polystyrene bead (diameter, 4 μm; specific gravity, 1.3), with a single to a few thick filaments of a molluscan smooth muscle attached, was made to slide along actin filament arrays (actin cables) in the internodal cell of an alga, mounted on the rotor of a centrifuge microscope. The bead moving along actin cables in the presence of ATP was subjected to centrifugal forces either opposite to the bead movement (positive loads) or in the same direction as the bead movement (negative loads). With positive loads increasing from zero to the maximum isometric force P 0, the velocity of bead movement decreased gradually to zero, exhibiting the hyperbolic force-velocity relation except for loads above 0.8 P 0. With further increase of positive loads above P 0, the bead was forced to move in the direction of centrifugal force, being eventually detached from actin cables at a load of ∼1.4 P 0. These features are very similar to the force-velocity relation in intact single muscle fibers. With negative loads increasing from zero to a certain value, on the other hand, the bead moved faster than the maximum unloaded velocity (1.5–3.5 μm/s, 20–25°C), until it was eventually detached from actin cables. These results indicate that our assay system is extremely promising for future combined biochemical and physiological studies on the mechanism of muscle contraction.

Architecture and function in the muscle sarcomere

Striated muscle sarcomeres in vertebrates comprise ordered arrays of actin and myosin filaments, organized by an elaborate protein scaffold. Recent innovative work in a number of laboratories has greatly improved our knowledge of these structures, their organization and their interactions. Structural details have been reported on myosin filaments, actin filaments, Z-bands, M-bands, titin, and nebulin. Time-resolved X-ray diffraction and electron microscopy are revealing the molecular movements involved in force production and regulation.

The Role of Actin Filaments in Patterning theCaenorhabditis elegansCuticle

Nematodes are covered by a cuticle with a prominent pattern of circumferentially oriented, parallel furrows. We report here that the pattern of furrows on the first larval cuticle ofCaenorhabditis elegans,which is secreted during embryogenesis, is coincident with a pattern of submembranous actin filament bundles in the epithelial cells that secrete the cuticle. We propose that the pattern of cortical actin filaments biases the growth of the epithelial cell membranes, creating a furrowed surface template for deposition of the first cuticle layer. This layer then detaches from the epithelial cell surface as additional, nonpatterned components of the cuticle are secreted. Furrows are present on the surfaces of each of the four larval cuticles inC. elegansand on the adult cuticle. We show that similar ordered arrays of actin filaments appear during each of the postembryonic molts when new cuticles are synthesized. Our analysis suggests that conditions or mutations that affect the pattern of cuticle furrows might cause primary defects in the cytoskeletal organization of the epithelial cells that secrete the cuticle.

A personal view of muscle and motility mechanisms.

This is a personal account of some of the successive steps in our understanding of the structural mechanism of muscle contraction during the last 45 years. It describes how I, as an ex-physicist, came to be studying muscle by X-ray diffraction in 1949; how the concepts of the double array of actin and myosin filaments and, later, the overlapping filament model and the sliding filament mechanism were developed; and how further electron microscope findings of the structural polarity of muscle filaments led to the suggestion that analogous structures and mechanisms might be involved in cellular motility. The article describes briefly how synchrotron radiation has made it possible to obtain detailed structural information about contracting muscle with millisecond time resolution and discusses some of the recent major advances in the field and the prospects of reaching a full understanding of the contraction mechanism.

Rearrangements of F‐actin during Stomatogenesis Visualised by Confocal Microscopy in Fixed and Permeabilised Tradescantia Leaf Epidermis

Abstract: New details of F‐actin organisation in leaf epidermal and stomatal cells were revealed by rhodamine — and fluorescein — phalloidin staining of fixed epidermal peels of Tradescantia virginiana and visualisation by confocal microscopy. Non‐specialised epidermal cells contain highly organised arrays of fine cortical actin filaments aligned in transverse or oblique orientations. In interphase guard mother cells (GMCs), the arrangement of cortical F‐actin changes on the periclinal and anticlinal cell walls at different times during differentiation. Initially, cortical F‐actin on the periclinal surfaces is oriented transversely and F‐actin is evenly distributed around the anticlinal walls. Following polarisation of the adjacent subsidiary mother cells (SMCs), actin in GMCs concentrates on the lateral anticlinal walls, but not on the transverse walls. Subsequently, F‐actin on the periclinal walls reorients to radial and then longitudinal. Organisation of F‐actin in SMCs appears to be influenced by the adjacent GMCs and co‐ordination in F‐actin arrangements in cells of the stomatal complex continues through to the formation of the guard cell pair. Our studies indicate that actin bands marking the division site in prophase cells, and detected in microinjected living material, are a particularly labile subset of F‐actin. Actin bands were difficult to preserve, even when aldehyde fixation was avoided, in contrast to all interphase and mitotic F‐actin.

Cytoskeletal reorganization of human platelets induced by the protein phosphatase 1/2 A inhibitors okadaic acid and calyculin A.

Okadaic acid (OA) and calyculin A (CLA), which are potent and specific inhibitors of serine/threonine protein phosphatases type 1 and 2A, have been shown to induce drastic changes in platelet morphology. The aim of this study was to analyse the molecular mechanisms of OA- or CLA-induced cytoskeletal reorganization, with a specific focus on microtubules and actin filaments. Confocal fluorescence microscopy revealed that OA or CLA altered the distribution of microtubules from marginal band arrangements to homogeneous patterns, consistent with the transmission-electron-microscopic finding that microtubules were fragmented and redistributed into pseudopod-like processes. In thrombin-activated platelets, OA or CLA induced extremely long pseudopods containing an array of microtubules and actin filaments, and a condensed mass of actin filaments in the centre of platelets. OA or CLA induced the constriction of actin filaments without an increase in filamentous (F)-actin, and also rather significantly inhibited actin polymerization in thrombin-activated platelets. Furthermore, neither OA or CLA enhanced phosphorylation of myosin light chain (MLC). By immunoprecipitation of platelet lysate with anti-alpha-tubulin antibody, a 90 kDa protein was co-precipitated with tubulin and was predominantly phosphorylated in the presence of OA. As the time-dependent phosphorylation of 90 kDa protein correlated well with the reorganization of microtubules, these data suggest that phosphorylation and dephosphorylation of this protein might play a role in the regulation of microtubule organization. These findings indicate that OA or CLA induces reorganization of microtubules and actin filaments via the phosphorylation of a microtubule-associated 90 kDa protein and an MLC-phosphorylation-independent mechanism. mechanism.