Fibroblast spreading in the presence of cytochalasin D: immunoelectron microscopy of the cytoskeleton

Spreading of mouse embryo fibroblasts in the presence of cytochalasin D (1 microgram/ml) was studied using scanning electron microscopy, immunofluorescence, and electron microscopy of platinum replicas. Whereas circular lamellae were formed around the cell body during normal spreading, separate processes appeared at the cell periphery during spreading in cytochalasin-containing medium. The processes gradually elongated and branched. Cytoskeletons of fibroblasts spreading in the cytochalasin-containing medium were obtained by Triton X-100 extraction. They contained microtubules, intermediate filaments, actin "paracrystals" looking like short microfilament bundles, and patches of a meshwork-granular material. Immunogold coating of the cytoskeletons with anti-actin antibody showed that some meshwork-granular patches were decorated with gold particles, whereas the others were not. Non-actin patches were usually located on the distal ends of the processes, thus leaving behind the actin cytoskeletal components during the process growth. Another characteristic feature of this unidentified material is its usual association with the substratum and microtubules. These results suggest that the process protrusion during cell spreading in cytochalasin-containing medium may occur not due to actin polymerization as in the control cells, but due to involvement of some other non-actin cytoskeletal components. These components seem to be able to move along microtubules and to bind to the substratum.     

Distribution of microtubules and other cytoskeletal filaments during myotube elongation as revealed by fluorescence microscopy

Summary Distribution of microtubules and other cytoskeletal filaments in growing skeletal muscle cells (myotubes) was studied in vitro by fluorescence microscopy using fluorescin-labeled antibodies and phalloidin, a specific antiactin drug. In the distal elongating tips of myotubes, microtubules were the major cytoskeletal elements; actin and intermediate filaments were much less abundant. On the other hand, colcemidand nocodozole-treatments caused disruption of microtubules and also prompt retraction of growth tips to form myosacs, a type of deformed myotube. Actin filaments remained unaffected during the retraction. The difference in the distribution of the 3 cytoskeletal filaments in the region of growth tips was most remarkable in the case of those myotubes in the process of recovery from myosacs. In an early phase of recovery, the cellular processes extending from myosacs were enriched with both microtubules and intermediate filaments, but not with actin filaments. Later, when the processes became further developed, intermediate filaments were scarce at the extreme ends. Fluorescein-labeled actin introduced by a micro-injection method was minimally incorporated into filaments in the cellular processes. We conclude that microtubules make up the cytoskeletal element which is most responsible for elongation or spreading of growth tips of myotubes in vitro.     

Microtubules may harden or soften cells, depending of the extent of cell distension

Experimental data show that disruption of microtubules causes cells to either become stiffer or softer. Current understanding of these behaviors is based on several different mechanisms, each of which can account for only stiffening or softening. In this study we offer a model that can explain both these features. The model is based on the cellular tensegrity idea. Key premises of the model are that cell shape stability is secured through pre-existing mechanical stress (prestress) borne by the actin cytoskeletal network, and that this prestress is partly balanced by cytoskeletal microtubules and partly by the extracellular matrix. Thus, disturbance of this balance would affect cell deformability. The model predicts that disruption of microtubules causes an increase or a decrease in cell stiffness, depending on the extent to which microtubules participate in balancing the prestress which, in turn, depends on the extent of cell spreading. In highly spread cells microtubules have a minor and negative contribution to cell stiffness, whereas in less spread cells their contribution is positive and substantial. Since in their natural habitat cells seldom exhibit highly spread forms, the above results suggest that the contribution of microtubules to cell deformability cannot be overlooked.     

Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.     

The roles of the cytoskeleton during cellulose deposition at the secondary cell wall

During secondary cell wall formation, developing xylem vessels deposit cellulose at specific sites on the plasma membrane. Bands of cortical microtubules mark these sites and are believed to somehow orientate the cellulose synthase complexes. We have used live cell imaging on intact roots of Arabidopsis to explore the relationship between the microtubules, actin and the cellulose synthase complex during secondary cell wall formation. The cellulose synthase complexes are seen to form bands beneath sites of secondary wall synthesis. We find that their maintenance at these sites is dependent upon underlying bundles of microtubules which localize the cellulose synthase complex (CSC) to the edges of developing cell wall thickenings. Thick actin cables run along the long axis of the cells. These cables are essential for the rapid trafficking of complex-containing organelles around the cell. The CSCs appear to be delivered directly to sites of secondary cell wall synthesis and it is likely that transverse actin may mark these sites.     

Complexity of the tensegrity structure for dynamic energy and force distribution of cytoskeleton during cell spreading

Cytoskeleton plays important roles in intracellular force equilibrium and extracellular force transmission from/to attaching substrate through focal adhesions (FAs). Numerical simulations of intracellular force distribution to describe dynamic cell behaviors are still limited. The tensegrity structure comprises tension-supporting cables and compression-supporting struts that represent the actin filament and microtubule respectively, and has many features consistent with living cells. To simulate the dynamics of intracellular force distribution and total stored energy during cell spreading, the present study employed different complexities of the tensegrity structures by using octahedron tensegrity (OT) and cuboctahedron tensegrity (COT). The spreading was simulated by assigning specific connection nodes for radial displacement and attachment to substrate to form FAs. The traction force on each FA was estimated by summarizing the force carried in sounding cytoskeletal elements. The OT structure consisted of 24 cables and 6 struts and had limitations soon after the beginning of spreading by declining energy stored in struts indicating the abolishment of compression in microtubules. The COT structure, double the amount of cables and struts than the OT structure, provided sufficient spreading area and expressed similar features with documented cell behaviors. The traction force pointed inward on peripheral FAs in the spread out COT structure. The complex structure in COT provided further investigation of various FA number during different spreading stages. Before the middle phase of spreading (half of maximum spreading area), cell attachment with 8 FAs obtained minimized cytoskeletal energy. The maximum number of 12 FAs in the COT structure was required to achieve further spreading. The stored energy in actin filaments increased as cells spread out, while the energy stored in microtubules increased at initial spreading, peaked in middle phase, and then declined as cells reached maximum spreading. The dynamic flows of energy in struts imply that microtubules contribute to structure stabilization.     

Dendrite-like process formation and cytoskeletal remodeling regulated by delta-catenin expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of delta-catenin (or NPRAP/Neurojungin), a member of p120(ctn) subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of delta-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. delta-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of delta-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When delta-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that delta-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Dendrite-like Process Formation and Cytoskeletal Remodeling Regulated by δ-Catenin Expression

Actin- and microtubule-mediated changes in cell shape are essential for many cellular activities. However, the molecular mechanisms underlying the interplay between the two are complex and remain obscure. Here we show that the expression of δ-catenin (or NPRAP/Neurojungin), a member of p120^ctn subfamily of armadillo proteins can induce the branching of dendrite-like processes in 3T3 cells and enhance dendritic morphogenesis in primary hippocampal neurons. This induction of branching phenotype involves initially the disruption of filamentous actin, and requires the growth of microtubules. The carboxyl-terminal truncation mutant of δ-catenin can cluster and redistribute the full-length protein, and dominantly inhibit its branching effect. δ-Catenin forms protein complexes and can bind directly to actin in vitro. The carboxyl-terminal truncation of δ-catenin does not interfere with its actin-binding capability; therefore the actin interaction alone is not sufficient for the induction of dendrite-like processes. When δ-catenin-transformed cells establish elaborate dendrite-like branches, the main cellular processes become stabilized and resist the disruption of both actin filaments and microtubules, as determined by fluorescent light microscopy and time-lapse recording analyses. We suggest that δ-catenin can effect a biphasic cytoskeletal remodeling event which differentially regulates actin and microtubules and promotes cellular morphogenesis.     

Behavior of mitochondria, microtubules, and actin in the triangular yeastTrigonopsis variabilis

Summary Dimorphic yeastTrigonopsis variabilis is a unique species that can form either an ellipsoidal or a triangular cell depending upon nutritional conditions. This fluorescence microscopic study was intended to correlate morphological changes of mitochondria in the triangular cells with the distribution of the cytoskeleton. In addition, unique features in the behavior of the cytoskeleton were also examined during triangular cell formation. In log-phase cells stained with 4,6-diamidino-2-phenylindole, mitochondrial nucleoids appeared as a string of beads throughout the vegetative growth. The profile of mitochondria stained by 3,3-dihexyloxacarbocyanine iodide showed a network corresponding to the fluorescence images of mitochondrial nucleoids in both mother and daughter cells. Cell-cycle-dependent fragmentation of mitochondria was not discerned. As the culture reached stationary phase, a network of mitochondria gradually changed to form unique rings that were located near the angles of triangular cells. When examined by immunofluorescence microscopy with anti-tubulin antibody, microtubules were found to be well developed along the sides of cells in the cytoplasm ofT. variabilis interphase cells. Although distributions of microtubules and mitochondria are different during cell cycle as a whole, cytoplasmic microtubules frequently extended along a part of the mitochondria in budded cells, suggesting correlation of microtubules and mitochondria. Rhodamine-phalloidin staining revealed both actin patches and cables. Actin cables elongated from mother cells into the buds and showed close proximity to mitochondria, although complete overlapping of both structures was rare. Moreover, actin patches localized on the mitochondrial network at a frequency of 65%. These results suggested that actin cables and patches, as well as microtubules, participated in the distribution of mitochondria. The localization of actin patches separated towards opposite ends at a bud tip when the bud grew to medium size. The unique localization of actin patches is responsible for bi-directional growth of the bud, forming triangular cells.     

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.     

Spreading of porcine kidney epithelial cells under normal conditions and under the effect of inhibitors of energy metabolism. II. Behavior of cellular organelles

In the present work the behavior of mitochondria and lysosomes during cell spreading has been investigated in normal conditions and under ATP-synthesis inhibitors: sodium aside and N,N-dicyclohexylcarbodiimide (DCCD). In the control culture, microtubules run along the stable edge and perpendicular to the leading edge in most of spreading cells. As a whole, microtubules form a dense network in these cells. However, the radial cells contain bundles of microtubules, radiating from the perinuclear area or form circular arrays around the nucleus. The microtubule network is more dense under inhibitory treatment, than in control conditions. In the control culture the spherical cells display numerous small mitochondria (staining with Rhodamine 123). In the process of cell spreading some elongated mitochondria appear, most of them being localized in the perinuclear area. The mitochondria of cells with radial microtubule organization are directed towards the cell periphery, while in cells with circular bundles of microtubules the mitochondria are localized chaotically. Under DCCD treatment the mitochondria retain the staining for 2-3 h. In the spreading cells, round mitochondria may be distributed all over the cytoplasm. In the presence of sodium aside the mitochondria are not stained. However, by means of phase contrast microscopy some disoriented thread-shaped structures are observed, obviously corresponding to mitochondria. In the control conditions, lysosomes (stained with Acridine orange) in spreading cells are dispersed chaotically, all over the cytoplasm, or are localized in the perinuclear area. In the presence of sodium aside lysosomes are observed only in the perinuclear area. Under DCCD treatment lysosomes do not accumulate the dye. Thus, the cytoskeleton modification and changes in the properties of membrane organelles, induced by ATP-synthesis inhibitors, do not prevent attachment, spreading or cell polarization.     

10 nm filaments in normal and transformed cells.

An antibody was raised against an electrophoretically homogeneous protein from cultured fibroblasts and shown to be directed against 10 nm filaments. The antiserum did not stain microtubules or actin microfilaments. The distribution of 10 nm filaments in normal cells was studied during growth, spreading, locomotion, mitosis, and after treatment with colchicine and cytochalasin B. The 58,000 dalton subunit protein is apparently all polymerized in the filaments which are insoluble in nonionic detergent. The distribution of 10 nm filaments is altered by colchicine treatments which disrupt microtubules. The organization of 10 nm filaments is altered in transformed cells.     

The mechanism of matrix vesicle formation. Studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation

The mechanism of matrix vesicle (MV) formation by growth plate chondrocytes in primary cell culture was assessed both by using drugs which interfere with assembly or disassembly of microfilaments and microtubules, as well as by comparison of the composition of chondrocyte microvilli with MV. Cytochalasin D, which is known to inhibit assembly of actin microfilaments, was found to stimulate the release of alkaline phosphatase-rich MV. This stimulatory effect was confirmed by studies with [3H]palmitate- and 32P-prelabeled cells which showed that cytochalasin D enhanced the release of labeled MV. In contrast, phalloidin, which blocks disassembly of microfilaments, suppressed release of cellular alkaline phosphatase into MV. The phospholipid composition of vesicles released by cells treated with cytochalasin D and phalloidin was virtually identical with that of the controls. In contrast, colchicine, which interferes with the assembly of microtubules, was found to cause fragmentation of the cells, producing large vesicles significantly different in lipid composition from MV. Microscopic studies revealed that cytochalasin D caused marked rounding and retraction of the cells, with evidence of actin withdrawal from the cell periphery. This led to cell surface blebbing and formation of small zeiotic bodies at the tips of cell processes. In contrast, phalloidin enhanced and stabilized the actin network within the cells. Chemical analysis of microvilli prepared from isolated chondrocytes revealed high levels of alkaline phosphatase and a phospholipid composition almost identical to MV. Electrophoretic profiles of microvillar proteins were again like that of MV, except for the presence of high levels of actin. This cytoskeletal protein was nondetectable in MV. Taken together with the effects of the drugs, the data indicate that cell surface microvilli are the precursors of MV and that retraction of the supporting microfilament network is essential for the release of these structures.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables

Microtubules and actin filaments interact and cooperate in many processes in eukaryotic cells, but the functional implications of such interactions are not well understood. In the yeast Saccharomyces cerevisiae, both cytoplasmic microtubules and actin filaments are needed for spindle orientation. In addition, this process requires the type V myosin protein Myo2, the microtubule end-binding protein Bim1, and Kar9. Here, we show that fusing Bim1 to the tail of the Myo2 is sufficient to orient spindles in the absence of Kar9, suggesting that the role of Kar9 is to link Myo2 to Bim1. In addition, we show that Myo2 localizes to the plus ends of cytoplasmic microtubules, and that the rate of movement of these cytoplasmic microtubules to the bud neck depends on the intrinsic velocity of Myo2 along actin filaments. These results support a model for spindle orientation in which a Myo2-Kar9-Bim1 complex transports microtubule ends along polarized actin cables. We also present data suggesting that a similar process plays a role in orienting cytoplasmic microtubules in mating yeast cells.     

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.     

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by β1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate β1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of β1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase C? bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of β1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cε is an essential regulatory step for the trafficking and activation of β1 integrins and cell spreading on collagen.     

Microtubules suppress blebbing and stimulate lamella extension in spreading fibroblasts

We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment, cells start blebbing, which continues for different times and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic way through stochastic formation of lamellipodia. A second mode that occurs in 15% of cells was rapid, isotropic spreading via formation of circular lamella. The rate of spreading was maximal at the beginning and decreased during the first hour according to a logarithmic law. After 60 min, many cells formed stable edges and started to migrate on the substrate. However, the cell area slowly continued to increase. Actin bundles were formed 20 min after cell attachment. They first run along the cell boundary. This system disassembled within 20–40 min and was substituted with stress fibers crossing the cell. In isotropically spread cells, no actin bundles were seen. Microtubules in the spreading cells enter into large blebs and all nascent lamellas; later, they form a radial array. When MTs have been depolymerized or stabilized blebbing started, before cells attach to the substrate and continue much longer than in control cells. After both treatments, the initial rate of spreading decreases several-fold and remains constant for many hours. After 24 h, the mean area occupied by cells with an altered MT system was the same as in control. Alteration of the MT system had a moderate effect on the actin system: formation of actin cables occurred at the same time as in control (within 20 min upon cell attachment); however, they started growing even in cells undergoing prolonged blebbing. Actin cables running along the cell margin were similar to those in control cells, but they did not disappear for up to 1 h. When stabilized, MTs form a chaotic array: they do not enter blebs and, in spread cells, run parallel to the cell margin at a distance of 3–5 μm. We conclude that dynamic MTs speed up completion of blebbing and promote early stages of fibroblast spreading.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.