On the role of microfilaments in cell-shape-mediated growth control of lens epithelial cells

With regard to the fact that, in anchorage-dependent lens epithelial cells, DNA synthesis can be switched on and off by cell flattening and cell rounding, respectively, the state of the microfilaments has been followed by labelling actin with FL-phalloidin during cell-shape alterations. Cell flattening proved to be accompanied by both a structural organization of actin filaments into stress fibres and an enlargement of the area of the cell nucleus. Cell rounding, on the other hand, caused the microfilament bundles to disappear and the area of the nucleus to become smaller. From the time course of the inhibition of DNA synthesis by cytochalasin B, it was inferred that functionally intact microfilaments are required for the entrance of the cells into DNA synthesis but not for the maintenance of ongoing DNA synthesis. The assumption has been made that the tension, generated by microfilaments during cell spreading, will affect the state of the plasma membrane as well as the shape and the structure of the nucleus, which in turn seems to be preparatory for cells to enter the cycle.     

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.     

Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae

In the present study, we demonstrate the actin cytoskeleton reorganization during nurse cells apoptosis of the olive fruit fly Dacus oleae. At the developmental stage 9A of oogenesis, the actin microfilaments are assembled in numerous ring canals and subcortically support all the nurse cells, as is shown by phalloidin-FITC staining. During the following stages, 9B and 10A, this structural pattern remains the same. The developmental stage 10B is characterized by actin microfilament rearrangement and formation of actin cables that are symmetrically organized around the nurse cell nuclei. At stage 11, when the dumping process begins, these actin cables seem to retain each nurse cell nucleus in the cell center, away from blocking the ring canals. The early stage 12 is characterized by an asynchronous nurse cell nuclear chromatin condensation, while at late stage 12 the actin cables become very thick, as adjacent ones overlap one another and traverse the disorganized apoptotic nurse cell nuclei that already have fragmented DNA, as is demonstrated by acridine orange staining and TUNEL assay. Finally, during stage 13, the apoptotic nuclear remnants are phagocytosed by the neighboring follicle cells. The data presented herein compared to previous reported results in Drosophila [Nezis et al., 2000: Eur J Cell Biol 79:610-620], demonstrate that actin cytoskeleton reorganization during nurse cell apoptosis is a developmentally regulated physiological mechanism, phylogenetically conserved in higher Dipteran.     

AIP1/WDR1 supports mitotic cell rounding

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.     

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.     

A study of the Influence of mevalonic acid and its metabolites on the morphology of swiss 3T3 cells

We used two model systems to investigate the effect of compactin, a competitive inhibitor of beta-hydroxy beta-methylglutarylcoenzyme A reductase, on the shape of Swiss 3T3 cells. We maintained cells in a quiescent state in medium deficient in platelet-derived growth factor (PDGF), or we added PDGF to quiescent cells to initiate traverse through a single cell cycle. In both systems, the cells responded to compactin by acquiring a characteristic rounded shape. Cell rounding seemed to depend on an induced deficiency of mevalonic acid (MVA) since the response could be prevented or reversed by adding MVA to the culture medium. Compactin-induced rounding appeared in PDGF-stimulated cells concomitantly with a compactin-mediated inhibition of DNA synthesis, and both effects had similar sensitivities to exogenous compactin and MVA. However, cell rounding seemed to be unrelated to other, previously observed effects of MVA deficiency. Compactin did not influence the total content of cell cholesterol, and little cholesterol was formed when we added radioactive MVA to round cells to effect shape change reversal. Measurement of the dolichol-dependent glycosylation of cell protein revealed no evidence of dolichol deficiency. In addition, reversal of cell rounding by MVA was not prevented by concentrations of tunicamycin that effectively blocked the incorporation of radioactive mannose into cell protein or by concentrations of cycloheximide that blocked protein synthesis. Taken together, our results suggest a new role for MVA or its products in the maintenance of cell shape.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期（即生殖细胞在形成后从靠壁部位移向中央部位的阶段）的水稻（Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程。在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成。微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状。尔后,中心微丝不断瞎长,最终在细胞中央的胞质内形成一个非常 类似多个纺锤体结合在一起的网络结构。这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质（或称周质）的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构。未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系。     

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.     

Finite-element analysis of the adhesion-cytoskeleton-nucleus mechanotransduction pathway during endothelial cell rounding: axisymmetric model

Endothelial cells possess a mechanical network connecting adhesions on the basal surface, the cytoskeleton, and the nucleus. Transmission of force at adhesions via this pathway can deform the nucleus, ultimately resulting in an alteration of gene expression and other cellular changes (mechanotransduction). Previously, we measured cell adhesion area and apparent nuclear stretch during endothelial cell rounding. Here, we reconstruct the stress map of the nucleus from the observed strains using finite-element modeling. To simulate the disruption of adhesions, we prescribe displacement boundary conditions at the basal surface of the axisymmetric model cell. We consider different scenarios of the cytoskeletal arrangement, and represent the cytoskeleton as either discrete fibers or as an effective homogeneous layer When the nucleus is in the initial (spread) state, cytoskeletal tension holds the nucleus in an elongated, ellipsoidal configuration. Loss of cytoskeletal tension during cell rounding is represented by reactive forces acting on the nucleus in the model. In our simulations of cell rounding, we found that, for both representations of the cytoskeleton, the loss of cytoskeletal tension contributed more to the observed nuclear deformation than passive properties. Since the simulations make no assumption about the heterogeneity of the nucleus, the stress components both within and on the surface of the nucleus were calculated. The nuclear stress map showed that the nucleus experiences stress on the order of magnitude that can be significant for the function of DNA molecules and chromatin fibers. This study of endothelial cell mechanobiology suggests the possibility that mechanotransduction could result, in part, from nuclear deformation, and may be relevant to angiogenesis, wound healing, and endothelial barrier dysfunction.     

Microfilament Orientation Constrains Vesicle Flow and Spatial Distribution in Growing Pollen Tubes

The dynamics of cellular organelles reveals important information about their functioning. The spatio-temporal movement patterns of vesicles in growing pollen tubes are controlled by the actin cytoskeleton. Vesicle flow is crucial for morphogenesis in these cells as it ensures targeted delivery of cell wall polysaccharides. Remarkably, the target region does not contain much filamentous actin. We model the vesicular trafficking in this area using as boundary conditions the expanding cell wall and the actin array forming the apical actin fringe. The shape of the fringe was obtained by imposing a steady state and constant polymerization rate of the actin filaments. Letting vesicle flux into and out of the apical region be determined by the orientation of the actin microfilaments and by exocytosis was sufficient to generate a flux that corresponds in magnitude and orientation to that observed experimentally. This model explains how the cytoplasmic streaming pattern in the apical region of the pollen tube can be generated without the presence of actin microfilaments.     

Relationships between cytoplasmic microtubular complex,DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age,serum deprivation,aphidicolin, cytochalasin B and colchicine)

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an “aging effect” upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).     

Relationships between cytoplasmic microtubular complex, DNA synthesis and cell morphology in mouse embryonic fibroblasts (effects of age, serum deprivation, aphidicolin, cytochalasin B and colchicine)

Aging, aphidicolin, serum deprivation and cytochalasin B induce a decrease in the rate of DNA synthesis, an increase in cell flattening (cell surface increase) and an extension of the cytoplasmic microtubular complex (CMTC). Age and experimental conditions affect the protein content of the cell, but there is no relationship between cell morphology and cell protein content. Serum deprivation, aphidicolin and cytochalasin B are more effective on DNA synthesis and cytoplasmic actin complex (CAC) of late than of early fibroblasts. Despite these facts, the cell morphology of late cells is fairly stable and is not affected by experimental conditions, which exert an "aging effect" upon the cell morphology in earlier cultures. Colchicine acts upon the CMTC, cell morphology and DNA synthesis at all ages of the cultures. It also induces disruption of the CAC, the intensity of the disruption depending on both the length of the treatment and the age of the culture: the sensitivity of the actin-microfilaments to colchicine increases with the mitotic age of the cells. We suggest that the microtubular integrity is needed, but not sufficient, to preserve the organization of the CAC into microfilaments. We propose a logical model comprising feedback loops between the number of the mitotic cycles, the rate of DNA synthesis, the extention rate of the plasma membranes and CMTC in normal fibroblasts. CMTC is associated, in this model, with the expression of negative or positive controls, depending on the grade of its extension (Fig. 9).     

Extracellular matrix- and cytoskeleton-dependent changes in cell shape and stiffness

Cell spreading is correlated with changes in important cell functions including DNA synthesis, motility, and differentiation. Spreading is accompanied by a complex reorganization of the cytoskeleton that can be related to changes in cell stiffness. While cytoskeletal organization and the resulting cell stiffness have been studied in motile cells such as fibroblasts, less is known of these events in nonmigratory, epithelial cells. Hence, we examined the relationship between cell function, spreading, and stiffness, as measured by atomic force microscopy. Cell stiffness increased with spreading on a high density of fibronectin (1000 ng/cm(2)) but remained low in cells that stayed rounded on a low fibronectin density (1 ng/cm(2)). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting f-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrate the relationship between cell stiffness and spreading in hepatocytes. They specifically show that normal actin and myosin function is required for hepatocyte spreading and DNA synthesis and demonstrate opposing effects on cell stiffness upon disruption of actin and myosin.     

The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol—the substrate for the first enzyme, namely P450_scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca²⁺/calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca²⁺/calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cylic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca²⁺/calmodulin possibly by redistributing cellular Ca²⁺. Ca²⁺/calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomoyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca²⁺/calmodulin) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.     

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

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

Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期(即生殖细胞在形成后从靠壁部位移向中央部位的阶段)的水稻(Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程.在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成.微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状.尔后,中心微丝不断延长,最终在细胞中央的胞质内形成一个非常复杂的类似多个纺锤体结合在一起的网络结构.这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质(或称周质)的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构.未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系.     

Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.     

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.     

Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilaments and microtubules in capillary endothelial cells

Capillary endothelial cells can be switched between growth and apoptosis by modulating their shape with the use of micropatterned adhesive islands. The present study was carried out to examine whether cytoskeletal filaments contribute to this response. Disruption of microfilaments or microtubules with the use of cytochalasin D or nocodazole, respectively, led to levels of apoptosis in capillary cells equivalent to that previously demonstrated by inducing cell rounding with the use of micropatterned culture surfaces containing small (<20 microm in diameter) circular adhesive islands coated with fibronectin. Simultaneous disruption of microfilaments and microtubules led to more pronounced cell rounding and to enhanced levels of apoptosis approaching that observed during anoikis in fully detached (suspended) cells, indicating that these two cytoskeletal filament systems can cooperate to promote cell survival. Western blot analysis revealed that the protein kinase Akt, which is known to be critical for control of cell survival became dephosphorylated during cell rounding induced by disruption of the cytoskeleton, and that this was accompanied by a decrease in bcl-2 expression as well as a subsequent increase in caspase activation. This ability of the cytoskeleton to control capillary endothelial cell survival may be important for understanding the relationship among extracellular matrix turnover, cell shape changes, and apoptosis during angiogenesis inhibition.