Roles of microfilaments in exocytosis: a new hypothesis

We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.     

Relationship between the presence of microfilaments and the cell cycle of HeLa cells in synchronus culture]

Some cytoplasmic organelles have showed characteristic variations which are related to the different cell cycle phases, in thymidine synchonized HeLa cells in culture. In these cells, the most modified organelles were intracytoplasmic membranes (endoplasmic reticulum) and microfilament arrangements. Microfilaments were numerous under the cell membrane, but also some of them were dispersed in dense bundles. These structures were seen around the nucleus, 12-14 h after removal of excess thymidine (G1). They migrated to the periphery of the cell during S and G2. During mitosis, they were directly under superficial membrane-associated microfilaments.     

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro

Summary The dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freezefracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with Factin in solution) in the circumferential/microf相似文献   

Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Cytoplasmic filaments in fetal and neonatal pig testis

Leydig cells in developing fetal pig testis contained during the fetal regressive phase large accumulations of intermediate filaments. Before and after this period these filaments were arranged in a criss-cross fashion. In the pig as well as in the dog testis these filaments have been characterized as vimentin. Within the vimentin aggregates occasionally a weak positive actin reaction was seen in pig but not in dog Leydig cells. Microfilaments were hardly observed. Most Sertoli cells contained a layer of actin microfilaments close to the basal cell membrane. In the lower cell compartment and around the nucleus (intermediate) vimentin filaments could be observed in a criss-cross configuration.     

A primary role for microfilaments, but not microtubules, in hormone-induced cytoplasmic retraction

The distributions of microfilaments and microtubules were studied during transient hormone-induced changes in cell shape (retraction-respreading). Two cell types (fibroblasts and bone cells), differentially responsive to parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂), were analysed. The cytoplasm of fibroblasts retracted in response to PGE₂ but not PTH, whereas bone cells could respond to both PGE₂ and PTH. Time-lapse photomicrography indicated that the retraction began within minutes of hormone addition, while respreading occurred over longer times, up to 8 h. Affinity-purified actin and tubulin antibodies were used to follow the appearance of microtubules and microfilaments during both the retraction and the respreading phases. Microtubules appeared not to reorganize noticeably, although they were squeezed closer together in cellular pseudopods; no extensive loss or growth was detectable. Microfilaments did alter drastically their appearance and distributions. Soon after hormone addition when earliest detectable cytoplasmic retraction was evident, microfilament bundles appeared to break down. Remaining microfilament bundles consisted of relatively short, non-aligned fragments or aggregates. During respreading, microfilament bundles regrew and realigned throughout the cytoplasm. These data suggest a primary role for microfilaments, but probably not microtubules, in these cell shape changes.     

Polarization of specific tropomyosin isoforms in gastrointestinal epithelial cells and their impact on CFTR at the apical surface

Microfilaments have been reported to be polarized in a number of cell types based both on function and isoform composition. There is evidence that microfilaments are involved in the movement of vesicles and the polarized delivery of proteins to specialized membrane domains. We have investigated the composition of actin microfilaments in gastrointestinal epithelial cells and their role in the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) into the apical membrane using cultured T84 cells as a model. We identified a specific population of microfilaments containing the tropomyosin (Tm) isoforms Tm5a and/or Tm5b, which are polarized in T84 cell monolayers. Polarization of this microfilament population occurs very rapidly in response to cell-cell and cell-substratum contact and is not inhibited by jasplakinolide, suggesting this involves the movement of intact filaments. Colocalization of Tm5a and/or Tm5b and CFTR was observed in long-term cultures. A reduction in Tm5a and Tm5b expression, induced using antisense oligonucleotides, resulted in an increase in both CFTR surface expression and chloride efflux in response to cAMP stimulation. We conclude that Tm isoforms Tm5a and/or Tm5b mark an apical population of microfilaments that can regulate the insertion and/or retention of CFTR into the plasma membrane.     

Modifications of microfilaments and microtubules induced by two hepatic tumor promoters,phenobarbital and biliverdin in non-transformed and transformed hepatic cell lines

Microfilaments and microtubules are components of the cytoskeleton which could be implicated in neoplastic transformation. We studied the effect of two hepatic tumor promoters, phenobarbital (PB) and biliverdin (BV), on microfilaments and microtubules of non-transformed (Cl₃) and transformed (FV) hepatic epithelial cells. Cl₃ non-transformed cells cultured in the presence of 1 × 10^–6M BV for 48 h showed a loss of F-actin, fragmentation of actin and the appearance of star-like structures in the cytoplasm, as well as loosening of the peripheral bundle of actin, and some ruffling of cell membranes. In Cl₃ cells exposed to 0.2 × 10^–3M PB a similar disappearance of F-actin staining and a very prominent ruffling of cell membrane were observed. BV and PB also produced in these cells modifications of microtubules characterized by a disappearance of centrosome staining in numerous cells, a condensed ring of tubulin around the nucleus and a depolymerized aspect of the microtubular network. All these modifications of microfilaments and microtubules closely resembled those observed in FV transformed cells in the absence of any treatment (Solvent DMSO only). We did not observe an effect of BV and PB on FV cells.The present data demonstrate that the cytoskeleton of non-transformed epithelial liver cells is sensitive to the action of liver tumor promoters suggesting that it might play a role as to yet be defined in the promotion mechanism.Abbreviations PB phenobarbital - BV biliverdin - TPA 12-0-tetradecanoyl-phorbol 13 acetate - GGT gamma-glutamyl-transpeptidase - DMSO dimethylsulfoxyde     

Disturbed structural interactions between microfilaments and tight junctions in rat hepatocytes during extrahepatic cholestasis induced by common bile duct ligation

 Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions. Accepted: 28 August 1996     

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells

Summary Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

An analysis of the role of microfilaments in the establishment and maintenance of asymmetry in Caenorhabditis elegans zygotes

Microfilaments are needed to generate asymmetry during the first cell cycle in Caenorhabditis elegans zygotes. To investigate when and how microfilaments participate in this process, we have "pulsed" zygotes with the microfilament inhibitor cytochalasin D (CD) at different times during the cell cycle. We have shown that microfilaments are only required during a narrow time interval approximately three-quarters of the way through the first cell cycle for the manifestations of asymmetry that occur during and subsequent to this interval. When CD treatment spans this critical time interval, pseudocleavage, pronuclear migration, germ-granule segregation (all of which occur during the interval), and movement of the mitotic spindle to an asymmetric position (which occurs later in the cell cycle) are perturbed. In contrast, embryos briefly treated with CD before or after the critical time interval manifest normal asymmetry. Our results suggest that in C. elegans microfilaments participate in the generation of zygotic asymmetry by providing spatial cues and/or serving as a part of the necessary machinery only during a brief period in the first cell cycle, and are not required to maintain asymmetries that have already been established.     

丁酸对体外培养的人鼻咽癌上皮细胞的影响

无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞     

Modeled microgravity causes changes in the cytoskeleton and focal adhesions, and decreases in migration in malignant human MCF-7 cells

Because cells are sensitive to mechanical forces, microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement, and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated in microgravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK, PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinβ1 and integrinβ4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.     

Cytokinesis in the C. elegans embryo: regulating contractile forces and a late role for the central spindle

Genetic and molecular studies in the nematode Caenorhabditis elegans have identified multiple essential pathways that regulate and execute cytokinesis in early embryonic cells. These pathways influence both the microfilament cytoskeleton and the microtubule cytoskeleton. Microfilaments are enriched throughout the cell cortex at all times during the cell cycle in embryonic cells. Cortical microfilaments are required for multiple processes in embryonic cells, including polar body extrusion during meiosis, anterior-posterior axis specification by the sperm-donated microtubule-organizing center, and cytokinesis during mitosis. In addition to contractile apparatus proteins that are required positively for cleavage furrow ingression, the Nedd8 ubiquitin-like protein modification pathway negatively regulates contractile forces outside the cleavage furrow during cytokinesis. Another pathway that acts positively during cytokinesis involves the mitotic spindle. The central spindle, where anti-parallel non-kinetochore microtubules overlap and are cross-linked, is required for a late step in cytokinesis, and other pathway(s) involved in membrane addition during cytokinesis may also require the central spindle. The amenability of C. elegans to classical genetics, the ease of reducing gene function with RNA interference, the completion of the genome sequence, and the availability of transgenic GFP fusion proteins that render the cytoskeleton fluorescent, all serve to make the early worm embryo an especially promising system for further advances in the identification of cytokinesis pathways, and in defining their interactions.     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

Impaired cell motility in chronic myeloid leukemic granulocytes related to altered cytoskeletal pattern

The bactericidal activity of polymorphonuclear leucocyte (PMNL) against infection stimulates cytoskeletal changes accompanied with alteration in adhesion and locomotion. Microfilaments, the motile apparatus is known to regulate these changes by polymerization of monomeric G-actin to fibrous F-actin. PMNL from chronic myeloid leukemia (CML) patients have been reported to be defective in locomotion in response to synthetic peptide, n-formyl-methionyl-leucyl-phenylalanine (fMLP) but the mechanism leading to defective locomotion and their spatial reorganization remains unclear. Therefore, in order to study the cause of defective motility of PMNL from CML patients the spatial distribution and reorganization of microfilaments and microtubules in response to fMLP have been examined by transmission electron (TEM) and scanning electron microscopy (SEM). Under SEM, the PMNL-CML surface appeared smoother with reduced ruffling resulting in rounding off cells with lesser polarized morphology. Unstimulated PMNL from normal as well as CML subjects showed shorter and fewer microtubules and evenly distributed microfilaments as compared to fMLP stimulated PMNL. It is proposed that the cause of defective locomotion was due to reduced surface activity as a consequence of altered cytoskeletal configuration. This phenomenon seems to be related to impaired functional appendages and as a whole led to the defective cell motility and hence reduced chemotaxis in PMNL from CML patients.