Banding and polarity of actin filaments in interphase and cleaving cells

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.     

Scanning Electron Microscopy of Cytoskeletal Elements in the Oral Apparatus of Tetrahymena1

ABSTRACT. Extraction of the ciliated protozoon Tetrahymena with nonionic detergents produces a surface-related cytoskeleton that consists of a basic lamina of whole-cell dimensions together with associated microtubule and microfilament systems, including all ciliary basal bodies. The organization of the isolated cytoskeleton has been studied using scanning electron microscopy, and several new features are described in the oral region. Here the ciliary basal bodies are arranged in a very stable and highly complex pattern. This pattern was found to be identical in the four species of Tetrahymena we examined. In addition, various microtubular bundles and two separate systems of filaments were observed in scanning electron micrographs of isolated oral skeletons. The appearance of the deep fiber bundle in preparations of this type suggests that it arises, at least in part, as an extension of the ribbed wall microtubules. On the basis of its distribution within the oral skeleton, one of the filament systems described is suggested to be a contractile system responsible for pinching off food vacuoles.     

Microfilament bundles: I. Formation with uniform polarity

The coelomocytes of the sea urchin, Strongylocentrotus droebachiensis, have been used as a model system to investigate the relative orientation of single actin-containing filaments to the cell membrane as they are regrouped in multifilament bundles during a cellular morphogenetic event. In detergent-treated, heavy meromyosin (HMM) incubated and negatively stained cells, the polarity of each microfilament, regardless of whether it occurs singly or in a bundle, is such that the arrowhead complexes formed along the length of each filament by the HMM decoration point inward away from the cell membrane and toward the center of the cell. A mechanism is proposed by which the uniformly polar bundles may be formed.     

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg²⁺ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.     

ABP50: An actin-binding elongation factor 1α from Dictyostelium discoideum

ABP50 is a polypeptide elongation factor 1α from Dictyostelium that is associated with the actin cytoskeleton. Upon chemotactic stimulation, ABP50 undergoes a dramatic cytoplasmic redistribution into newly formed surface projections and in vitro binds to and bundles actin filaments. Many questions are raised by this interaction pertaining to the spatiotemporal regulation of protein synthesis and cytoskeletal organization by extracellular signals.     

Identification of Novel Graded Polarity Actin Filament Bundles in Locomoting Heart Fibroblasts: Implications for the Generation of Motile Force

We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25–2.5 μm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 μm, but can reach up to about 30 μm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward.

By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body.

This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.

     

Heavy meromyosin induces sliding movements between antiparallel actin filaments

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Novel Mode of Cooperative Binding between Myosin and Mg-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca²⁺-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg²⁺-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg²⁺-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.     

The Interaction of Mip-90 with Microtubules and Actin Filaments in Human Fibroblasts

The novel microtubule-interacting protein Mip-90 was originally isolated from HeLa cells by using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on β-tubulin. Biochemical and immunocytochemical data have suggested that the association of Mip-90 with the microtubule system contributes to its cellular organization. Here we report the interaction patterns of Mip-90 with microtubules and actin filaments in interphase human fibroblasts. A polyclonal monospecific antibody against Mip-90 was used for immunofluorescence microscopy analysis to compare the distribution patterns of this protein with tubulin and actin. A detailed observation of fibroblasts revealed the colocalization of Mip-90 with microtubules and actin filaments. These studies were complemented with experiments using cytoskeleton-disrupting drugs which showed that colocalization patterns of Mip-90 with microtubules and actin filaments requires the integrity of these cytoskeletal components. Interestingly, a colocalization of Mip-90 with actin at the leading edge of fibroblasts grown under subconfluency was observed, suggesting that Mip-90 could play a role in actin organization, particularly at this cellular domain. Mip-90 interaction with actin polymers was further supportedin vitroby cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constantK_a= 1 × 10⁶M⁻¹, while an stoichiometry Mip-90/actin of 1:12 mol/mol was calculated. Western blots of the immunoprecipitates revealed that Mip-90 associated to both actin and tubulin in fibroblasts extracts. These studies indicate that Mip-90, described as a microtubule-interacting protein, also bears the capacity to interact with the microfilament network, suggesting that it may play a role in modulating the interactions between these cytoskeletal filaments in nonneuronal cells.     

Actin in Tetrahymena paravorax: Ultrastructural Localization of HMM-Binding Filaments in Glycerinated Cells1,2

Actin has been identified in the ciliated protozoon Tetrahymena paravorax on the basis of the ultrastructural detection of filaments typically decorated with heavy meromyosin (HMM) in glycerinated microstome cells. These filaments are widely distributed in endoplasmic and cortical regions and can form bundles. They are particularly numerous in elongating cells; HMM-binding filaments run approximately parallel to rib microtubules in the ectoplasm of the right wall of the buccal cavity and seem to extend to the cytopharyngeal region, suggesting some role of actin in maintenance of the crest-trough pattern of ribbed wall and/or in formation of food vacuoles. Extensive actin bundles are observed below some membranellar areas and are thought to follow the course of the microtubular “deep fiber bundle.” The “fine filamentous reticulum” underlying the oral ribs and the “apical ring” extending beneath kinetosomes of ciliary couplets display filaments that do not bind HMM and are ? 14 nm in diameter. No evidence for actin in these structures was obtained in the present study. The “specialized cytoplasm” of the cytostome-cytopharyngeal region appears as an undecorated reticulum with 20 nm-spaced nodes. Occasionally HMM-binding filaments were found inside the macronucleus, just beneath its envelope. Actin is suggested to be involved in cell shaping and in control of the transport of food vacuoles.     

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of (i) microfilament bundles containing proteins of contractile structures; (ii) microtubules; and (iii) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).     

Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells

After trypsinization and replating, BHK-21 cells spread and change shape from a rounded to a fibroblastic form. Time-lapse movies of spreading cells reveal that organelles are redistributed by saltatory movements from a juxtanuclear position into the expanding regions of cytoplasm. Bidirectional saltations are seen along the long axes of fully spread cells. As the spreading process progresses, the pattern of saltatory movements changes and the average speed of saltations increases from 1.7 MICROMETER/S during the early stages of spreading to 2.3 micrometer/s in fully spread cells. Correlative electron microscope studies indicate that the patterns of saltatory movements that lead to the redistribution of organelles during spreading are closely related to changes in the degree of assembly, organization, and distribution of microtubules and 10-nm filaments. Colchicine (10 microgram/ml of culture medium) reversibly disassembles the microtubule-10-nm filament complexes which form during cell spreading. This treatment results in the disappearance of microtubules and the appearance of a juxtanuclear accumulation of 10-nm filaments. These changes closely parallel an inhibition of saltatory movements. Within 30 min after the addition of the colchicine, pseudopod-like extensions form rapidly at the cell periphery, and adjacent organelles are seen to stream into them. The pseudopods contain extensive arrays of actinlike microfilament bundles which bind skeletal-muscle heavy meromyosin (HMM). Therefore, in the presence of colchicine, intracellular movements are altered from a normal saltatory pattern into a pattern reminiscent of the type of cytoplasmic streaming seen in amoeboid organisms. The streaming may reflect either the activity or the contractility of submembranous microfilament bundles. Streaming activity is not seen in cells containing well-organized microtubule-10-nm filament complexes.     

Actin and tubulin in teratocarcinoma cells. Amount and intracellular organization upon cytodifferentiation.

Embryonal carcinoma (EC) cells and differentiated derivatives grown in tissue culture have rather similar amounts of actin and tubulin. Indirect immunofluorescent microscopy with antibodies to actin shows striking differences in the actin organization in the different teratocarcinoma derivatives. In the EC cells, actin is found predominantly in ruffles and in surface protrusions, as well as in the cytoplasm, but microfilament bundles are not seen. Some of the differentiated clones contain strongly stained microfilament bundles; others contain actin arrangements which appear to be characteristic of the particular cell type. Indirect immunofluorescence microscopy with antibody to tubulin suggests that cytoplasmic microtubules are present both in the EC cells and in the various differentiated states studied. However, the ease with which microtubules can be documented is dependent on how cells are spread on the substratum. During in vitro differentiation of EC cells, changing patterns of actin distribution appear. Cells at the edge of the colony show the characteristic changes in microfilament and microtubular organization before those in the center.     

High-voltage electron microscopy of whole,critical-point dried plant cells

Summary The lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plant's normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical cortical pattern of microtubules which appears to play no part in chloroplast motility.Abbreviations EDTA ethylenediaminetetra-acetic acid - EM electron microscopy - FITC fluorescein-iso-thiocyanate - HVEM high voltage electron microscopy - PIPES piperazine-NN-bis-2-ethanesulphonic acid     

Embryogenesis in <Emphasis Type="Italic">Sedum acre</Emphasis> L.: structural and immunocytochemical aspects of suspensor development

The changes in the formation of both the actin and the microtubular cytoskeleton during the differentiation of the embryo-suspensor in Sedum acre were studied in comparison with the development of the embryo-proper. The presence and distribution of the cytoskeletal elements were examined ultrastructurally and with the light microscope using immunolabelling and rhodamine-phalloidin staining. At the globular stage of embryo development extensive array of actin filaments is present in the cytoplasm of basal cell, the microfilament bundles generally run parallel to the long axis of basal cell and pass in close to the nucleus. Microtubules form irregular bundles in the cytoplasm of the basal cell. A strongly fluorescent densely packed microtubules are present in the cytoplasmic layer adjacent to the wall separating the basal cell from the first layer of the chalazal suspensor cells. At the heart-stage of embryo development, in the basal cell, extremely dense arrays of actin materials are located near the micropylar and chalazal end of the cell. At this stage of basal cell formation, numerous actin filaments congregate around the nucleus. In the fully differentiated basal cell and micropylar haustorium, the tubulin cytoskeleton forms a dense prominent network composed of numerous cross-linked filaments. In the distal region of the basal cell, a distinct microtubular cytoskeleton with numerous microtubules is observed in the cytoplasmic layer adjacent to the wall, separating the basal cell from the first layer of the chalazal suspensor cells. The role of cytoskeleton during the development of the suspensor in S. acre is discussed.     

Distribution of microfilament bundles during rotation of the nucleus in 3T3 cells treated with monensin

Cytoskeletal aspects of monensin-treated 3T3 cells with rotating nuclei were studied by immunofluorescence. The pattern of intermediate filaments and microtubules appeared unchanged when compared with control cells having a stationary nucleus. In contrast, the actin microfilament bundles appeared to have a consistent distribution in cells with rotating nuclei. Typically, we did not find long microfilament bundles that traverse the length of the cytoplasm of cells that were fixed at the time of nuclear rotation. Instead, there was a local distribution of short microfilament bundles situated ventrally to the nucleus and oriented at various angles to one another and to the predominant distribution of microfilament bundles in the cell. The observations suggest that the actin cytoskeleton is reorganized locally before or during rotation of the nucleus.     

Microtubule disassembly enhances reversible cytochalasin-dependent disruption of actin bundles in characean internodes

Summary Parallel bundles of actin filaments at the cortex-endoplasm interface provide tracks for myosin-generated cytoplasmic streaming in characean internodes. These bundles resist disassembly or structural modification when exposed to 10 μM cytochalasin D (CD) even though this concentration of CD rapidly (within minutes) but reversibly arrests streaming. Unexpectedly, we discovered that prolonged treatment with lower concentrations of CD could partially disassemble the subcortical actin bundles. Actin bundles became discontinuous following one- to several-day treatment with concentrations (6 μM) that reduced but did not arrest streaming, and the residual fragments mostly remained parallel to the chloroplast files. When microtubules were concurrently disassembled with tubulin-specific drugs, however, low CD concentrations (2.5–3 μM) completely arrested bulk streaming, disrupted the largely 2-dimensional actin bundle array and caused the formation of a coarse, thick-meshed actin network that extended from the cortex to the endoplasm. Despite such massive reconstruction, drug removal enabled cells to recover continuous parallel bundles and streaming. Recovery was possible if both or just one of the drugs were removed. In recovered cells, the streaming pattern frequently redeveloped in new directions that did not follow the chloroplast files, and later, chloroplast files readjusted to the new polarity established by the actin bundles. This first report on the complete and reversible disassembly of characean actin bundles provides new insights into the mechanism of actin bundle assembly and organization and supports the idea of indirect interactions between actin filaments and microtubules.     

State of actin during the cycle of cohesiveness of the cytoplasm in parthenogenetically activated sea urchin egg

By the DNase I inhibition assay it is shown that the cytoplasmic matrix isolated 60 min after procaine activation of Paracentrotus lividus eggs contains about 20% of the total egg actin, mostly in polymerized form (85%). Electron microscopy studies on this cytoplasmic structure after treatment with heavy meromyosin (HMM), reveal that the decorated actin filaments are organized in bundles which are distributed radially, with the arrowheads pointing towards the central region. In addition few microtubules and a network of non-decorated microfilaments of about 3 nm diameter are observed. From the cytoplasmic pH determination and the DNase I inhibition assay on homogenates of eggs which were taken at different times of activation, it cannot be inferred that a direct relationship between the increase in the cytoplasmic pH and the increase in the amount of polymerized actin or of cytoplasmic matrix exists. Activation experiments carried out in the presence of colchicine shows that, although the formation of the cytoplasmic matrix is inhibited, polymerization of actin still occurs. Moreover, from the inhibition effects of cytochalasin B (CB) added before the activator it is shown that polymerization of actin is a necessary step for the organization of the cytoplasmic matrix. However, the cycles of cohesiveness of the cytoplasm observed in the course of the activation process do not appear to depend on cycles of polymerization and depolymerization of actin.