Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

On the relation between distinct components of the cytoskeleton: an epitope shared by intermediate filaments, microfilaments and cytoplasmic foci.

Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.     

Organization of stress fibers in cultured fibroblasts after extraction of actin with bovine brain gelsolin-like protein

Mouse and quail embryo fibroblasts were extracted with Triton X-100 and the resulting cytoskeletons were treated with gelsolin-like actin-capping protein (the 90-kDa protein-actin complex isolated from bovine brain). Staining of cells with rhodamine-conjugated phalloin or an antibody to actin did not reveal any actin-containing structures after treatment with the 90-kDa protein-actin complex. Extraction of actin was confirmed by SDS-gel electrophoresis. Immunofluorescence microscopy showed that vinculin and α-actinin were released from the cytoskeletons together with actin. However, myosin remained associated with the cytoskeleton after treatment with the 90-kDa protein-actin complex. The distribution of myosin in treated cells showed no significant difference from that in control cells: in both cases myosin was localized mainly in the stress fibers. Double-fluorescence staining showed the absence of actin in myosin-containing stress fibers of treated cells. The ultrastructural organization of actin-depleted stress fibers was studied by transmission electron microscopy of platinum replicas. On electron micrographs these fibers appeared as bundles of filaments containing clusters of globular material. It is concluded that myosin localization in stress fibers does not depend on actin.     

A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton

The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.     

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.     

Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin

We investigated the effects of calcitonin (CT) and parathyroid hormone (PTH) on the distribution of actin, tubulin, vimentin, and on cell size in cultured chick osteoclasts. In addition, we studied the effects of colchicine on intracellular acidity. Osteoclasts were isolated from the endosteum of 2-3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with CT for 30 min or PTH for 60 min and were observed after immunocytochemical staining of cytoskeletal proteins. In untreated cells, actin was found in both a filamentous and a punctate staining pattern, with indented or invaginated regions free of punctate spots. The tubulin distribution in untreated cells was characterized by a pattern of microtubules radiating from the cell center and running parallel to the cell edge. Vimentin staining was usually localized to the perinuclear area. There were no changes in cytoskeletal element distribution or morphology attributable to PTH treatment. Osteoclasts treated with CT were more irregularly shaped, contained more retraction fibers, and were more rounded, with a denser array of cytoskeletal elements in the cell center. In addition, the mean area of the CT-treated cells was significantly less than that of the untreated cells. The actin distribution after CT treatment was still characterized by both a filamentous and a punctate pattern. After CT treatment, vimentin staining appeared more centrally localized than in untreated cells and tubulin staining revealed microtubules which now extended to the retracted cell margin. These results indicate that isolated osteoclasts respond to CT by significant morphological changes which are reflected in the distribution of the major cytoskeletal elements. Disruption of the microtubular system by colchicine treatment also resulted in an initial increase in intracellular acidity, suggesting the involvement of microtubules in the movement of acid-laden vesicles to the exterior.     

Immunohistochemistry of chaetognath body wall muscles

Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (～80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (～10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (～6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.     

Studies on the organization and localization of actin and myosin in neurons

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.     

Immunolocalization of the intermediate filament-associated protein plectin at focal contacts and actin stress fibers.

The distribution of plectin in the cytoplasm of Rat1 and glioma C6 cells was examined using a combination of double and triple immunofluorescence microscopy and interference reflection microscopy. In cells examined shortly after subcultivation (less than 48 h), filamentous networks of plectin structures, resembling and partially colocalizing with vimentin filaments, were observed as reported in previous studies. In cells kept attached to the substrate without growth for periods of 72 h to 8 days (stationary cultures), thick fibrillary plectin structures were observed. These structures were located at the end of actin filament bundles and showed co-distribution with adhesion plaques (focal contacts), vinculin, and vimentin. Only relatively large adhesion plaques (dash-like contacts) were decorated by antibodies to plectin, smaller dot-like contacts at the cell edges remained undecorated. Moreover, in stationary Rat1 cells plectin structures were found to be predominantly colocalized with actin stress fibers. However, after treatment of such cells with colcemid, plectin's distribution changed dramatically. The protein was no longer associated with actin structures, but was distributed diffusely throughout the cytoplasm. After a similar treatment with cytochalasin B, plectin's association with stress fibers again was completely abolished, although stress fibers were still present. The association of plectin with focal contact-associated intermediate filaments was demonstrated also by immunogold electron microscopy of quick-frozen, deep-etched replicas of rat embryo fibroblasts. These data confirm previous reports suggesting a relationship between intermediate filaments on the one hand, and actin stress fibers and their associated plasma membrane junctional complexes, on the other. Furthermore, the data establish plectin as a novel component of focal contact complexes and suggest that plectin plays a role as mediator between intermediate filaments and actin filaments.     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

The distribution of actin in cultured ovarian granulosa cells

Ovarian granulosa cells grown on glass coverslips were split by a "sandwich" technique. Using this technique we describe a complex filamentous network in the cytoplasm of cultured granulosa cells that was composed of a branching and anastomosing lattice of filaments 20-40 nm in diameter. Since filament identification is impossible on the basis of size, split cells were decorated with S-1 fragments of rabbit skeletal muscle myosin. It was readily apparent that the major constituent of the filamentous lattice was actin. Actin was organized in large bundles in which individual filaments were longitudinally aligned. Actin was also observed organized in a loose network throughout the remainder of the cytoplasm. Actin appeared to be intimately associated with organelle and plasma membranes. Coated pits were also a site of actin-filament interaction. Filament polarity was generally away from the membrane with which filaments were associated.     

Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin- actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.     

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.     

Myofibrillogenesis in skeletal muscle cells in the presence of taxol

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.     

The molecular organization of myosin in stress fibers of cultured cells

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.     

Three-dimensional distributions of desmin and vimentin in cultured hamster cardiomyocytes using the immunogold deep-etching replica technique

The distributions of desmin and vimentin intermediate filaments in cultured hamster heart cells were examined by immunofluorescent microscopy and an immunogold deep-etching replica technique in combination with electron microscopy. Fluorescent studies showed the overall staining patterns of the myocytes as well as the fibroblasts. Monoclonal antibodies (Da, D₃) to desmin showed punctate staining for the myocytes, while polyclonal desmin (pD) stained in a filamentous pattern. Fibroblasts stained strongly with monoclonal anti-vimentin (Va), but did not stain with the desmin probes. Deep-etched immunogold studies confirmed at the ultrastructural level that monoclonal anti-desmin antibodies stain individual intermediate filaments in an intermittent pattern. Monoclonal (D₃) antibody stained the intermediate filaments heavily and continuously at the cell peripheries, while it stained intermittently in the cell body, similar to the Da monoclonal. Monoclonal anti-vimentin stained only intermediate filaments in fibroblasts. Our studies show a heterogeneity of staining within the cultured heart cells when various anti-desmin and anti-vimentin antibodies are used.     

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the chemical coding of each class of ICs.