Significance of the cytoskeleton for cytoplasmic organization and cell organelle dynamics in epithelial cells of fresh-water sponges

Summary The cytoskeleton in epithelial cells ofSpongilla lacustris is constructed of microtubules radiating from the nuclear region and terminating at the cell periphery as well as microfilaments forming a cortical layer beneath the plasma membrane and distinct fibers in the cytoplasmic matrix. Single frame analysis and in vivo labeling of mitochondria with Rh 123, endosomes or lysosomes with TRITC-BSA, endoplasmic reticulum (ER) with DiOC₆ (3) and dictyosomes with C₆-NBD-ceramide points to the microtubular system as a candidate for controlled cytoplasmic organization and active transport of these cell organelles. In epithelial cells with an intact microtubular system, mitochondria and endosomes or lysosomes show a regular shuttle movement between the nucleus and the cell periphery with a velocity of 1.3–1.4 m/s; the ER forms a more or less dynamic two-dimensional network in the entire cytoplasmic matrix, and dictyosomes are arranged in a ring-like manner around the nucleus. In epithelial cells treated with colchicine or colcemid, mitochondria and endosomes or lysosomes gather in the perinuclear region and become immobile; the ER accumulates near the cell center, whereas most dictyosomes distribute randomly over the whole cytoplasm. Transformation of the microfilament system with cytochalasin D has no influence on cell organelle distribution and dynamics but impedes cell locomotion and cell surface activities.Abbreviations BSA bovine serum albumin - C₆-NBD-ceramide 6-[(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)caproyl]sphingosine - DiOC₆(3) 3,3-dihydroxyloxacarbocyanine jodide - DMSO dimethylsulfoxide - EGTA ethylenediaminetetraacetic acid - GTX glycerol-Triton-X-100 - PBS phosphate buffered saline - PEG polyethylene glycol - PIPES 1,4-piperazine-N,N-bis-(2-ethanesulfomc) acid - Rh 123 rhodamine 123 - TRITC tetramethylrhodamine isothiocyanate     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Cytoskeletal control of the apical surface transformation of rat uterine epithelium

Summary— During early pregnancy, in the lead up to blastocyst implantation, the apical cell surface of luminal epithelial cells of the rat uterus undergo a dramatic shape transformation. This study aims to investigate the role of the cytoskeleton in this apical transformation by considering the effects of the drugs cytochalasin D and colchicine on the uterine luminal cell surface. The results are determined using transmission and scanning electron microscopy. In vivo exposure to cytochalasin D during oestrus, as well as on day 1 of pregnancy, did not affect the long, regular surface microvilli. This drug, however, did disrupt the terminal web within the apical cytoplasm of these cells. Disruption of microfilament (MF) polymerization by cytochalasin D on day 4 of pregnancy induced a cell surface transformation, resulting in the appearance of numerous irregular projections normally present during blastocyst implantation on day 6 of pregnancy. Colchicine did not alter the uterine microvilli of oestrus or day 1 pregnant tissue. Unlike the effect of cytochalasin D, colchicine-induced microtubule (MT) disruption on day 4 of pregnancy did not increase irregular projections and hence this treatment did not result in the cell surface appearance associated with blastocyst implantation. These results indicate that the disruption of MF, rather than MT, contributes to the transformation of the uterine luminal cell surface during the lead up to blastocyst attachment.     

Reorganization of plasma membrane-associated 36 000 dalton protein upon drug-induced redistribution of cytokeratin

Epithelial PtK2 cells were used as a model to study the possible redistribution of the major tyrosine protein kinase substrate, p36, upon drug-induced reorganization of the cytokeratin network. Cells were grown on glass coverslips and exposed to cytochalasin D (CD), colcemid or a combination of the two. The cytokeratin type intermediate filaments of the cells were redistributed by treatment of the cells with colchicine and CD. Simultaneous changes in cytokeratin and in p36 antigen were observed by double-label immunofluorescence. In control PtK2 cells, p36 was distributed characteristically at the cortical cytoplasm. Neither colchicine nor CD alone was able to cause a major reorganization of cytokeratin or p36. Their combined effect resulted in formation of blebs containing abundant p36 at the cell surface and at cell-cell junctions. Actin, on the other hand, was reorganized to similar configurations by CD alone. These observations show for the first time a drug-induced redistribution of p36. The results suggest a relationship between membrane-associated p36 and the cytoskeletal fibres that terminate at the plasma membrane.     

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.     

Complementary roles of microtubules and microfilaments in the lung fibroblast-mediated contraction of collagen gels: Dynamics and the influence of cell density

Summary Fibroblasts are important cellular components in wound healing, scar formation, and fibrotic disorders; and the fibroblast-populated collagen-gel (FPCG) model allows examination of fibroblast behavior in an in vitro three-dimensional environment similar to that in vivo. Contraction of free-floating FPCGs depends, on an active and dynamic cytoskeleton, and the contraction dynamics are highly influenced by cell density. We investigated mechanistic differences between high- and low-cell density FPCG contraction by evaluating contraction dynamics in detail, using specific cytoskeletal disruptors. Collagen gels were seeded with human lung fibroblasts at either high (HD) or low (LD) density, and incubated with or without cytoskeletal disruptors colchicine (microtubules) or cytochalasin D (microfilaments). Gel area was measured daily. FPCG contraction curves were essentially sigmoidal, featuring an initial period of no contraction (lag phase), followed by a period of rapid contraction (log phase). Contraction curves of HD-FPCGs were distinct from those of LD-FPCGs. For example, HD-FPCGs had a negligible lag phase (compared with 3 d for LD-FPCGs) and exhibited a higher rate of log-phase contraction. Both colchicine and cytochalasin dose-dependently inhibited contraction but specifically affected different phases of contraction in HD- and LD-FPCGs; and colchicine inhibited LD-FPCGs much more than HD-FPCGs. The data indicate that LD- and HD-FPCGs contract through different primary mechanisms. Microtubules and microfilaments are both complementarily and dynamically involved in the contraction of FPCGs, and cell density influences primary cytoskeletal mechanisms. These results provide valuable information about fibroblast behavior in healing and fibrosis, and may suggest novel treatment options.     

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.     

Effect of colchicine, vinblastine, and cytochalasin B on cell surface anionic sites of Tritrichomonas foetus

Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic site-containing macromolecules located on the cell surface of T. foetus.     

Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

Microtubule-disrupting drugs (nocodazole, colchicine) and cytochalasin D, which inhibits the polymerization of the actin microfilaments, were used to study the role of the cytoskeleton in protein secretion in the polarized Madin-Darby canine kidney (MDCK) epithelial cells. Two proteins were analyzed. The gp 80 glycoprotein complex, which in untreated cells is sorted into the apical pathway and lysozyme, which is released randomly at both cell surfaces in transfected MDCK cells. Our results show that cytochalasin D has no influence on the transport of the gp 80 complex and lysozyme to either cell surface. However, in the presence of nocodazole or colchicine the secretion of both proteins at the apical cell surface is reduced by 50% with a concomitant increase in the basolateral release. These data suggest that microtubules are necessary for an efficient secretion of proteins at the apical cell surface of MDCK cells. In regard to the yet unresolved discrepancy concerning the involvement of microtubules in the transport of membrane proteins to the apical surface of MDCK cells, our results are consistent with the data of Rindler et al. (Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J. Cell. Biol. 104, 231-241) who observed a nonpolarized delivery of the influenza virus hemagglutinin in the presence of nocodazole or colchicine.     

The cytoskeleton and rat granulosa cell steroidogenesis: possible involvement of microtubules and microfilaments

The participation of both microtubules and microfilaments in granulosa cell steroidogenesis was assessed by monitoring the effects of colchicine (0-250 microM) and/or cytochalasin B (0-10 micrograms/ml) or dihydrocytochalasin B (0-2.0 micrograms/ml) on cellular morphology and production of progestins during 24 h of culture. Both colchicine and the cytochalasins increased granulosa cell production of progesterone and of 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-OH-progesterone) in a dose-dependent manner. The largest increase in steroidogenesis (about 2- to 3-fold) was observed at 4-250 microM colchicine and at 2-10 micrograms/ml cytochalasin. Those concentrations of the inhibitors of microtubule or microfilament polymerization that stimulated basal progestin production also markedly influenced cell spreading. Whereas cells cultured for 24 h in medium alone became very flattened with numerous cytoplasmic extensions, those cultured with colchicine (0.2-250 microM) or cytochalasin (0.4-2 micrograms/ml) were much less spread and progressively became more rounded and regular in outline. These changes in cell morphology were reflected by decreases in the mean area occupied by the cells on the culture surface of up to 60-65% and reductions in mean contour index values from 5.7 +/- 0.1 (control) to 3.9 +/- 0.1 (250 microM colchicine), 4.2 +/- 0.1 (2 micrograms/ml cytochalasin B), or 4.1 +/- 0.1 (2 micrograms/ml dihydrocytochalasin B). Cultures containing both colchicine and cytochalasin B exhibited a greater steroidogenic response than that elicited by either inhibitor alone. For example, granulosa cell progesterone production was stimulated almost 2-fold by 4 microM colchicine or 2 microM/ml cytochalasin B, but 5.5-fold by 4 microM colchicine plus 2 micrograms/ml cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cytoskeletal perturbant drugs on ecto 5''-nucleotidase, a concanavalin A receptor

Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A (Con A) have been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma cells treated with microfilament- and microtubule-perturbing drugs. By scanning electron microscopy MAT-C1 cells exhibit a highly irregular surface, covered with microvilli extending as branched structures from the cell body. MAT-A, MAT-B, and MAT-B1 cells have a more normal appearance, with unbranched microvilli, ruffles, ridges, and blebs associated closely with the cell body. MAT-C cells have an intermediate morphology. Treatment of MAT-A, MAT-B, or MAT-B1 cells with Con A causes rapid redistribution of Con A receptors. Both cytochalasins and colchicine cause alternations in the receptor redistributions. Receptors on MAT-C1 cells are highly resistant to redistribution, even in the presence of cytoskeletal perturbant drugs. The cooperativity of the inhibition of AMPase by Con A was investigated in MAT-A and MAT-C1 cells. Untreated cells exhibit no cooperativity. If either subline is treated with colchicine, cytochalasin B or D, or dibucaine, cooperativity is observed. Lumicolchicine has no effect. Theophylline or dibutyryl cyclic AMP prevents the effects of either colchicine or cytochalasin. The concentration required for half-maximal induction of cooperativity is 0.3--0.4 microM for both colchicine and cytochalasin D, which is in the appropriate range for specific microtubule and microfilament disruptions. The effectiveness of the cytochalasins (E greater than D greater than B) is consistent with their known effects on microfilaments. No direct correlation was observed between the induction of cooperativity and drug-induced changes in Con A receptor redistribution or cell morphology. The morphology of MAT-A cells is grossly altered by cytochalasins or dibucaine and somewhat less by colchicine. MAT-C1 cells exhibit more minor alterations in morphology as a result of these drug treatments. The results of this study indicate that the inhibition of AMPase, which is a Con A receptor, is a different process from the redistribution of the bulk of the Con A receptors, possibly short range membrane interactions rather than global effects on the cell.     

Organization of the endoplasmic reticulum in dividing cells of the gymnosperms Pinus brutia and Pinus nigra,and of the pterophyte Asplenium nidus

Endoplasmic reticulum (ER) organization in the dividing cells of the pterophyte Asplenium nidus and of the gymnosperms Pinus brutia and Pinus nigra has been studied by immunolocalization techniques using the monoclonal antibody 2E7, which recognizes luminar ER resident proteins containing C-terminal HDEL sequences. In the pterophyte, the ER reorganization during cell cycle is similar to that in angiosperms. Among others, prominent ER gatherings were found at the mitotic spindle poles and in the phragmoplast during cytokinesis. However, in the gymnosperms examined, the ER displays a unique pattern of reorganization not described so far. In both the Pinus species, well-defined ER patterns are successively formed during cell cycle. They are the preprophase ER-band, the prophase- metaphase- and anaphase ER-spindle, the interzonal ER-system, the ER-phragmoplast and an ER-system lining the daughter cell wall. The ER patterns are closely similar to that of the correspondent microtubule (MT) arrangements with which they are co-organized. Observations made on P. nigra root-cells affected by oryzalin, colchicine and cytochalasin D favour the conclusion that the pattern of ER organization is controlled during mitosis and cytokinesis by the MT cytoskeleton.     

Microtubule disassembly delays the G2-M transition in vertebrates

When cell cultures in growth are treated with drugs that cause microtubules to disassemble, the mitotic index (MI) progressively increases as the cells accumulate in a C-mitosis. For many cell types, however, including rat kangaroo kidney PtK(1) cells, the MI does not increase during the first several hours of treatment [1-3] (Figure 1). This 'lag' implies either that cells are entering mitosis but rapidly escaping the block, or that they are delayed from entering division. To differentiate between these possibilities, we fixed PtK(1) cultures 0, 90 and 270 minutes after treatment with nocodazole, colcemid, lumi-colcemid, taxol or cytochalasin D. After 90 minutes, we found that the numbers of prophase cells in cultures treated with nocodazole or colcemid were reduced by approximately 80% relative to cultures treated with lumi-colcemid, cytochalasin D or taxol. Thus, destroying microtubules delays late G(2 )cells from entering prophase and, as the MI does not increase during this time, existing prophase cells do not enter prometaphase. When mid-prophase cells were treated with nocodazole, the majority (70%) decondensed their chromosomes and returned to G(2) before re-entering and completing prophase 3-10 hours later. Thus, a pathway exists in vertebrates that delays the G(2)-M transition when microtubules are disassembled during the terminal stages of G(2). As this pathway induces mid-prophase cells to transiently decondense their chromosomes, it is likely that it downregulates the cyclin A-cyclin-dependent kinase 2 (CDK2) complex, which is required in vertebrates for the early stages of prophase [4].     

Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity

We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization.     

Factors which disorganize microtubules or microfilaments increase the frequency of cell transformation by polyoma virus.

Griseofulvin, 12-O-tetradecanoyl phorbol-13-acetate, melittin, epidermal growth factor, vinblastine, cytochalasin B, podophyllotoxin, colcemid, and colchicine were unable to transform cells but could increase from 8- to 40-fold the frequency of cell transformation by polyoma virus. The 3T3-like cells were resting at confluence and were exposed to the drug only during the 1st week after viral infection. Griseofulvin, a tumor promoter, reduced or increased the frequency of transformation depending on the dose with which the infected cells were treated. The antitumor activity of tumor promoters is discussed.     

Regulation of the subcellular distribution and gene expression of GABA(A) receptor by microtubules and microfilaments in cultured brain neurons

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABA(A)R alpha(1) or alpha(2) mRNA was present in 92 +/- 2% or 94 +/- 2% of the cytochalasin D-treated neurons, both of which were higher than 71 +/- 2-74 +/- 3% of the control and colchicine-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.     

The effects of cytochalasin B and colchicine on the morphogenesis of mitochondria in Drosophila hydei during meiosis and early spermiogenesis

Summary Morphogenesis of mitochondria in male germ cells in cultivated cytocysts begins in early prophase I at which time mitochondria thicken and become ordered along the spindle apparatus during meiosis. At the end of the second meiotic division they aggregate to form the Nebenkern.In the presence of colchicine or cytochalasin B mitochondria are able to begin differentiation, although the correct course of meiosis is not guaranteed. In medium supplemented with colchicine they undergo normal thickening but do not aggregate, in a pattern known from untreated cultures. This may indicate that microtubules are involved in the aggregation process of mitochondria as colchicine is known to inhibit microtubule formation. Moreover, in cell cultures treated with cytochalasin B mitochondrial aggregation does occur; it is concluded that microfilaments, which are sensitive to cytochalasin B, do not play a detectable role in the aggregation of mitochondria.     

COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.