Organelle redistribution during PK cell spreading in normal conditions and in the presence of sodium azide

Cell detachment from solid substratum results in a changed arrangement of cytoplasmic organelles and, primarily, in disruption of the centrosome and Golgi complex colocalization. In the course of cell spreading, reorganization of intracellular compartments occurs by stages. At first, mitochondria disperse in the cytoplasm volume, then the centrosome and Golgi complex unite, and finally collapse of intermediate filaments disappears. In the presence of Na azide the establishment of intracellular compartments during cell spreading proceeds in a similar manner. However, unlike the normal conditions, here atypical intracellular compartments appear: some of these containing lysosomes and lipid inclusions and located in the perinuclear area, and others with extended cisterns of endoplasmic reticulum on the cell periphery. Addiitionally, in the presence of Na azide the density of microtubules and intermediate filaments increases. It has been suggested that changes in the cytoskeleton organization lead to an enhanced cell spreading.     

Effect of acrylamide on the cytoskeleton and apoptosis of bovine lens epithelial cells

Acrylamide, a known disrupter of intermediate filaments, has been used to produce the collapse of vimentin filaments in bovine lens epithelial (BEL) cells, and its potential modulation of staurosporine-induced apoptosis has been investigated. In BEL cells, short treatments with acrylamide caused the collapse of vimentin filaments and microtubules and the almost complete disappearance of stress fibers, with thick f-actin bundles remaining in the cell periphery. Actin organization was less affected in cells pretreated with colchicine and in spreading cells, suggesting that extended microtubules and vimentin filaments are required for acrylamide to produce its maximal effects. Acrylamide alone slightly increased apoptosis compared to controls. However, simultaneous exposure to acrylamide and staurosporine for 8h produced significantly less apoptosis than staurosporine alone, and preincubation with acrylamide followed by staurosporine markedly reduced apoptosis at 8 and 24h of treatment. Acrylamide seems therefore to have a dual effect on BEL cell survival.     

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

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

Role of the cytoskeleton in the morphological normalization of transformed cells in culture

The role of the cytoskeleton in morphological normalization of transformed cells was studied. Mouse cells of the L197/6 clonal line were fused by polyethylene glycol and replated. The multinuclear cells were more spread than control ones: the ratio of the cell-occupied area to the number of the nuclei increased 2-3 times as a result of multinucleation. Instead of the spindle-like morphology typical for control cells they became star-like with larger lamellar regions located between radially oriented cell processes. According to the immunofluorescent data these processes contained thick bundles of microtubules and intermediate filaments. Destruction of these bundles with colcemide led to a decrease in the area occupied by multinuclear cells but did not change significantly the area occupied by control cells. The role of microtubules and intermediate filaments in cell spreading is discussed.     

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

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

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Reorganization of endothelial cells cytoskeleton during formation of functional monolayer in vitro

Endothelium lining the inner surface of vessels regulates permeability of vascular wall by providing exchange between blood circulation in vessels and tissue fluid and therefore performs a barrier function. Endothelial cells (ECs) in culture are able to maintain the barrier function peculiar to cells of vascular endothelium in vivo. The endothelial monolayer in vitro is a unique model system that allows studying interaction of cytoskeletal and adhesive structures of endotheliocytes from the earliest stages of its formation. In the present work, we described and quantitatively characterized the changes of EC cytoskeleton from the moment of spreading of endotheliocytes on glass and the formation of the first contacts between neighbor cells until formation of a functional confluent monolayer. The main type of intermediate filaments of ECs are vimentin filaments. At different stages of endothelial monolayer formation, disposition of vimentin filaments and their amount do not change essentially, they occupy more than 80% of the cell area. Actin filaments system of endotheliocytes is represented by cortical actin at the cell periphery and by bundles of actin stress fibers organized in parallel. With formation of contacts between cells in native endothelial cells, the number of actin filaments rises and thickness of their bundles increases. With formation of endothelial monolayer, there are also changes in the microtubules system—their number increases at the cell edge. At all stages of EC monolayer formation, the number of microtubules in the region of the already formed intercellular contacts exceeds the number of microtubules in the free lamella region of the cell.     

Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells

After trypsinization and replating, BHK-21 cells spread and change shape from a rounded to a fibroblastic form. Time-lapse movies of spreading cells reveal that organelles are redistributed by saltatory movements from a juxtanuclear position into the expanding regions of cytoplasm. Bidirectional saltations are seen along the long axes of fully spread cells. As the spreading process progresses, the pattern of saltatory movements changes and the average speed of saltations increases from 1.7 MICROMETER/S during the early stages of spreading to 2.3 micrometer/s in fully spread cells. Correlative electron microscope studies indicate that the patterns of saltatory movements that lead to the redistribution of organelles during spreading are closely related to changes in the degree of assembly, organization, and distribution of microtubules and 10-nm filaments. Colchicine (10 microgram/ml of culture medium) reversibly disassembles the microtubule-10-nm filament complexes which form during cell spreading. This treatment results in the disappearance of microtubules and the appearance of a juxtanuclear accumulation of 10-nm filaments. These changes closely parallel an inhibition of saltatory movements. Within 30 min after the addition of the colchicine, pseudopod-like extensions form rapidly at the cell periphery, and adjacent organelles are seen to stream into them. The pseudopods contain extensive arrays of actinlike microfilament bundles which bind skeletal-muscle heavy meromyosin (HMM). Therefore, in the presence of colchicine, intracellular movements are altered from a normal saltatory pattern into a pattern reminiscent of the type of cytoplasmic streaming seen in amoeboid organisms. The streaming may reflect either the activity or the contractility of submembranous microfilament bundles. Streaming activity is not seen in cells containing well-organized microtubule-10-nm filament complexes.     

Associations of elements of the Golgi apparatus with microtubules

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.     

Endothelial microtubules as integral part of the mechanism controlling the level of stimulated secretion of van Willebrand factor

We used double immunofluorescence and electron microscopy to study the spatial relationships between Weibel--Palade bodies (WPBs) and cytoskeletal elements in endothelial cells treated with thrombin or cytoskeleton-damaging agents. We have found that some WPBs undergo translocation towards the centrosome in 5 min in the cells treated with thrombin, cytochalasin B or calyculin A. The cells treated with thrombin or cytochalasin exhibit depletion of WPBs, whereas WPBs found at the cell periphery were colocalized with intermediate filaments. There was a precise colocalization observed between the WPBs and microtubules in the calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse by demecolcine, intermediate filaments and WPBs both translocated to the perinuclear region. The data provide the first direct evidence that secretory granules utilize microtubules to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is dependent on their interaction with vimentin filaments.     

Microtubules and the endoplasmic reticulum are highly interdependent structures

The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system.     

Structural interaction of cytoskeletal components

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament “branches” are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the “microtrabecular network” (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).     

The cytoskeleton of spreadingDictyostelium amoebae

Summary The cytoarchitectural elements ofDictyostelium discoideum amoeba have been visualized by light and electron microscopy in cells prepared with mixtures of glutaraldehyde and Triton-X-100. After negative staining, the peripheral regions of spreading amoebae show a complex meshwork of actin filaments, the majority of which were less than 0.25 microns in length. Multiple branch points, end to side abutments and cross-overs were characteristic features of the actin meshworks. Filopodia extending from the cell periphery consisted of bundles of actin filaments that penetrated into and merged with the actin meshworks in the spreading lamellae. Microtubules emanating from the nucleus associated body penetrated to differing extents into the actin meshworks, sometimes extending close to the cell periphery.Dictyostelium cytoskeletons preparted as described here should prove useful for further studies on the locomotory mechanism.     

The Sertoli cell in lizards

Sertoli cells of lizards are characterized by variable size, abundant smooth and rough endoplasmic reticulum, multivesicular bodies, lipid vacuoles probably related to the spermatogenic cycle, and mitochondria of normal size. The cytoskeleton contains actin, particularly abundant in the cell periphery, vimentin all around the nucleus and throughout the rest of the cytoplasm. Moreover, microtubules are distributed in the cell periphery. The junctional complexes demonstrate the presence of a very efficient blood-testis barrier, containing tight, gap, tight-gap, septate-like, desmosome-like, and "Sertoli-Sertoli" junctions. In the last, the actin layer interposed between the plasma membrane and the subsurface cistern is absent. The desmosome-like junctions are surrounded by 7-nm filaments and not by intermediate filaments.     

Structure and composition of the cytoskeleton of nucleated erythrocytes: III. Organization of the cytoskeleton of Bufo marinus erythrocytes as revealed by freeze-dried platinum-carbon replicas and immunofluorescence microscopy

Platinum-carbon (Pt-C) replicas of freeze-dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule-containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross-bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface-associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt-C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.     

Coalignment of microtubules, cytokeratin intermediate filaments, and collagen fibrils in a collagen-secreting cell system

The distribution of microtubules and intermediate filaments in the collagen-secreting scleroblasts of the goldfish scale was investigated by immunofluorescence and electron microscopy. Many of the microtubules and cytokeratin type intermediate filaments formed bundles that were aligned with the underlying, parallel collagen fibrils. The intermediate filament bundles were evenly spaced and located adjacent to the basal plasma membrane. The microtubules, on the other hand, were located further away from the membrane, although many were found very close to the intermediate filament bundles. No detectable change was observed in scleroblast microtubules when cells on scales were treated with colchicine or cooled (greater than or equal to 0 degrees C) for up to 1 h. Cells had to be cooled overnight before the microtubules were affected. The final number and length of the microtubules in the cell depended only on the final steady-state temperature and not the temperature history of the scale cell, and steady state was reached more slowly at colder temperatures. The microtubules but not the intermediate filaments rapidly (within 5 min) and reversibly depolymerized when cells were chilled to -2 approximately -4 degrees C. When chilled cells were warmed, the microtubules polymerized back, within 15 min at room temperature, to the same pattern of parallel coalignment with the underlying collagen. They appeared to repolymerize via two different pathways: (1) a radial growth outwards from the microtubule organizing center followed by a progressive realignment with the underlying collagen and (2) a gradual and simultaneous polymerization along cold-stable, antitubulin staining fibers. These fibers were also aligned with the collagen fibrils and may be related to the aligned intermediate filaments.     

Ultrastructure of the centrosome in L cells

Three types of microtubule-organizing centers are present in the interphase L-cells: centriolar matrix, pericentriolar satellites, and electron-dense bodies that are not attached to the centrioles. Different types of microtubule-organizing centers may be present simultaneously in the same centrosome. In most of the cells some microtubules have their proximal ends free, rather than attached to the microtubule-organizing center. A network of intermediate filaments is condensed around the centrosome. The intermediate filaments run from the centrosome parallel to the microtubules. Although the filaments are often in close proximity to the centrioles and microtubules, direct contacts between them are rare. The intermediate filaments have convergence foci of their own in the centrosome.     

10 nm filaments in normal and transformed cells.

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