Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Cytoskeleton in TFG-beta- and bFGF-modulated endothelial monolayer repair

Endothelial cell proliferation and migration in vitro is depressed by transforming growth factor beta (TFG-beta) and enhanced by basic fibroblast growth factor (bFGF) treatment. This study examines interactions between cytoskeletal changes and cell proliferation in regenerating endothelial monolayers treated with bFGF, TFG-beta, and both factors. As previously described by others, monolayer regeneration is enhanced by bFGF and reduced by TFG-beta. Endothelial cell morphology is altered by TFG-beta treatment. Cells lose their cobblestone appearance and assume a pleomorphic shape. Actin microfilament staining is modified in both intact and regenerating TFG-beta-treated monolayers as well. There is a loss of dense peripheral band staining and an enhancement in staining intensity of cytoplasmic stress fibers. No such alterations are seen in bFGF-treated cultures. Cell proliferation at the wound edge, as indicated by bromodeoxyuridine incorporation, is inhibited by TGF-beta. Although monolayer repair is modulated by growth factor treatment, centrosome reorientation and microtubule staining patterns are not altered by either factor. Thus these factors appear to have effects on a mechanism(s) other than centrosome reorientation which may be involved in repair of denuded endothelial monolayers.     

Modulation of Ca2(+)-dependent intercellular adhesion in bovine aortic and human umbilical vein endothelial cells by heparin-binding growth factors

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that growth of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic fibroblast growth factor (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic fibroblast growth factor (aFGF, 6 ng/ml) or endothelial cell growth supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding growth factors (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in growing cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at 22 hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.     

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.     

Role of cytoskeleton in gravisensing of the root elongation zone in Arabidopsis thaliana plants

In order to reveal the involvement of tubulin microtubules and actin microfilaments in gravisensing reactions in the distal elongation zone of root, Arabidopsis thaliana plants stably transformed with MAP4-GFP construct were grown under slow clinorotation. Experiments have shown that stabilization of cell growth in the distal elongation zone of Arabidopsis seedling root is provided by common structural organization of microtubules and microfilaments, and interrelations between microtubules and microfilaments is highly dependent upon the type of cell differential growth. Less pronounced effect of microfilament disruption on microtubule organization has been observed under clinorotation and it suggests the existence of complex mechanism of cooperation between microtubules and microfilaments which is probably, masked on earth.     

Understanding the role of the cytoskeleton in the complex regulation of the endothelial repair

Actin microfilaments and microtubules are important cytoskeletal proteins that regulate endothelial repair through alterations in cell shape and through regulation of cell migration following wounding of the endothelium. Upstream pathways have been identified in the regulation of actin and microtubule organization, especially small GTPases. Recently, there have been numerous proteins suggested to be capable of regulating interaction between microtubules and microfilaments to mediate microtubule regulation of endothelial repair, an important process in limiting injury to the artery wall and in reducing the extent of arterial disease. If disrupted, a rapid repair mechanism is important in reestablishing the integrity of the endothelium in order to reestablish its function as a macromolecular barrier, a thromboresistant surface, and a biologically active tissue. Strategies to improve repair should alter the pathobiology of the atherosclerotic plaque and thus improve the prognosis of patients with atherosclerosis.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.     

Hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in Arabidopsis thaliana

Elongation of diffusely expanding plant cells is thought to be mainly under the control of cortical microtubules. Drug treatments that disrupt actin microfilaments, however, can reduce elongation and induce radial swelling. To understand how microfilaments assist growth anisotropy, we explored their functional interactions with microtubules by measuring how microtubule disruption affects the sensitivity of cells to microfilament-targeted drugs. We assessed the sensitivity to actin-targeted drugs by measuring the lengths and diameters of expanding roots and by analysing microtubule and microfilament patterns in the temperature-sensitive Arabidopsis thaliana mutant microtubule organization 1 (mor1-1), along with other mutants that constitutively alter microtubule arrays. At the restrictive temperature of mor1-1, root expansion was hypersensitive to the microfilament-disrupting drugs latrunculin B and cytochalasin D, while immunofluorescence microscopy showed that low doses of latrunculin B exacerbated microtubule disruption. Root expansion studies also showed that the botero and spiral1 mutants were hypersensitive to latrunculin B. Hypersensitivity to actin-targeted drugs is a direct consequence of altered microtubule polymer status, demonstrating that cross-talk between microfilaments and microtubules is critical for regulating anisotropic cell expansion.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.     

白芨萜类化合物对人脐静脉内皮细胞凋亡和细胞骨架的作用

利用白芨萜类化合物处理人脐静脉内皮细胞（HUVECs）并进一步研究了细胞凋亡及细胞骨架.白芨萜类化合物可拮抗血管内皮细胞生长因子（VEGF）和碱性成纤维细胞生物因子（bFGF）刺激的HUVECs增殖并诱导细胞凋亡.在处理的HUVECs中caspase-8活性明显增加.流式细胞术分析显示经处理的HUVECs的凋亡率随处理时间延长而升高.通过对微管进行免疫荧光染色和微丝进行荧光染色后,用激光共焦扫描显微观察表明,白芨萜类化合物处理的HUVECs中的微管和微丝发生改变甚至被破坏.因此,白芨萜类化合物造成HUVECs凋亡很可能是通过促使微管解体以及微丝去组装造成的.     

Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of platinum replicas

We have studied the distribution of cytoskeletal elements in detergent-extracted mouse embryo fibroblasts using the platinum replica technique. It was shown that lamelloplasm can be subdivided into three zones: 1) the ruffle edge with dense microfilament meshwork; 2) the sparse zone adjacent to the ruffle edge and containing relatively few cytoskeletal elements; 3) the lamella proper occupied with a three-dimensional network of microfilaments, microtubules, intermediate filaments; this zone contained adhesion plaques corresponding to cell-substrate focal contacts and associated with the microfilament bundle ends. The cytoskeleton structure of the central (endoplasm) region of the cell was markedly different from that of the lamelloplasm. Its main feature was a dense microfilament sheath at the dorsal cell surface. Sites of microfilament bundle convergence can be visualized near the nucleus after partial removal of the sheath by more complete detergent extraction.     

Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization

Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis.     

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 m?g/ml) and colchicine (2 × 10^?5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re-tightening” following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process. © 1995 Wiley-Liss, Inc.     

The Type II Arabidopsis Formin14 Interacts with Microtubules and Microfilaments to Regulate Cell Division

Formins have long been known to regulate microfilaments but have also recently been shown to associate with microtubules. In this study, Arabidopsis thaliana FORMIN14 (AFH14), a type II formin, was found to regulate both microtubule and microfilament arrays. AFH14 expressed in BY-2 cells was shown to decorate preprophase bands, spindles, and phragmoplasts and to induce coalignment of microtubules with microfilaments. These effects perturbed the process of cell division. Localization of AFH14 to microtubule-based structures was confirmed in Arabidopsis suspension cells. Knockdown of AFH14 in mitotic cells altered interactions between microtubules and microfilaments, resulting in the formation of an abnormal mitotic apparatus. In Arabidopsis afh14 T-DNA insertion mutants, microtubule arrays displayed abnormalities during the meiosis-associated process of microspore formation, which corresponded to altered phenotypes during tetrad formation. In vitro biochemical experiments showed that AFH14 bound directly to either microtubules or microfilaments and that the FH2 domain was essential for cytoskeleton binding and bundling. However, in the presence of both microtubules and microfilaments, AFH14 promoted interactions between microtubules and microfilaments. These results demonstrate that AFH14 is a unique plant formin that functions as a linking protein between microtubules and microfilaments and thus plays important roles in the process of plant cell division.     

The reorganization of microfilaments, centrosomes, and microtubules during in vitro small wound reendothelialization

The repair of small endothelial wounds is an important process by which endothelial cells maintain endothelial integrity. An in vitro wound model system was used in which precise wounds were made in a confluent endothelial monolayer. The repair process was observed by time-lapse cinemicrophotography. Using fluorescence and immunofluorescence microscopy, the cellular morphological events were correlated with the localization and distribution of actin microfilament bundles and vinculin plaques, and centrosomes and their associated microtubules. Single to four-cell wounds underwent closure by cell spreading while wounds seven to nine cells in size closed by initially spreading which was then followed at approximately 1 h after wounding by cell migration. These two processes showed different cytoskeletal patterns. Cell spreading occurred independent of centrosome location. However, centrosome redistribution to the front of the cell occurred as the cells began to elongate and migrate. While the peripheral actin microfilament bundles (i.e., the dense peripheral band) remained intact during cell spreading, they broke down during migration and were associated with a reduction in peripheral vinculin plaque staining. Thus, the major events characterizing the closure of endothelial wounds were precise in nature, followed a specific sequence, and were associated with specific cytoskeletal patterns which most likely were important in maintaining directionality of migration and reducing the adhesion of the cells to their neighbors within the monolayer.     

Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.     

Role of the cytoskeleton during injury-induced cell migration in corneal endothelium

The role of microfilaments and microtubules during injury-induced cell migration of corneal endothelial cells in situ along their natural basement membrane has been investigated using organ culture. In the noninjured tissue, actin is localized at or near the plasma membrane, whereas tubulin is observed as a delicate lattice pattern throughout the cytoplasm. Twenty-four hours after a circular freeze injury, cells surrounding the wound area extend processes into this region. Fluorescent microscopy using phallotoxins and anti-tubulin antibodies demonstrated the presence of stress fibers and microtubule reorganization within these cells. Between 24 and 48 h post-injury endothelial cells move into the wound region, and by 48 h, the injury zone is repopulated and the monolayer is becoming reestablished. When injured corneas are placed in media containing 5 x 10(-7) M cytochalasin B, endothelial cell migration occurs; but it is slow, and wound closure is not complete even by 72 h. In contrast, when tissues are cultured in the presence of 10(-8) M colchicine, cell movement is greatly reduced, complete wound closure does not occur, and endothelial cells at the wound edge fail to display extensions typical of migrating cells. Furthermore, when injured endothelia are exposed to 0.05 micrograms/ml of actinomycin D for 15 min within the first hour after injury and transferred back into culture media lacking the drug for the duration of the experiment, migration does not occur and the wound persists. These actinomycin D treated cells remain viable as shown by their ability to incorporate 3H-uridine and 3H-thymidine. Fluorescence microscopy of actinomycin D treated tissues revealed the presence of stress filaments but disorganized microtubule patterns. Interestingly, 24 h after injury, if the tissue is exposed to actinomycin D, even for periods of up to 1 h, migration is not inhibited. Our results indicate that injury-induced endothelial cell movement appears to be more dependent on microtubule than microfilament reorganization and may require a critical timing of macromolecular synthesis.     

FLUORESCENT LABELLING OF THE CYTOSKELETON IN CERAMIUM STRICTUM (RHODOPHYTA)1,2

Using rhodamine-phalloidin to detect F-actin/microfilaments and indirect immunofluorescence to detect tubulin/microtubules, we studied the cytoskeleton in axial cells of Ceramium strictum Harv., especially microfilaments and microtubules associated with cytoplasmic strands (trabeculae) that extend longitudinally through the central vacuole. As axial cells attained mature size, trabeculae became progressively thinner, branched, and then broke down. An extensive microfilament array was present in peripheral parts of axial cells as well as in trabeculae. Microfilament arrays were highly disrupted by cytochalasin-B; this resulted in small irregular actin structures in axial cell peripheries and occasional dense aggregations at the base of cells. No actin-fluorescence was detected in intact trabeculae after cytochalasin-B treatment. Microtubules formed a primary structural component in trabeculae, which were disrupted by griseofulvin (5 to 0.005 μM) but reformed after two days in griseofulvin-free medium.