Substratum attachment of embryonic mesoderm cells in culture

Summary The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by inteference reflection microscopy and transmission electron microscoy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrate, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were pedominant. By contrast, when the cells were immobile, after 5 d in cultue, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts. This work was supported by grants from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.     

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cellsubstratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell’s underside by the thin lamellae and filopodia of a cell in close apposition.     

Amoeboid locomotion of Acanthamoeba castellanii with special reference to cell-substratum interactions

The amoeboid locomotion of Acanthamoeba castellanii has been studied by observation of individual cells moving on a planar glass substratum. Cell-substratum interactions involved in traction have been observed by reflexion interference microscopy. A variable part of the ventral surface of A. castellanii formed a protean platform, the 'associated contact', from which filopodia were subtended; these established stable, focal adhesions (approximately 0.4 micron diameter) on the substratum beneath. Surprisingly, acanthopodia, a prominent feature of this protozoon, did not play an obvious role in traction. The dimensions of the cell-substratum gap in the associated contact could be modulated by the concentration of ambient electrolyte. Dilution of electrolyte from 50 mM-KC1 to 2mM resulted in (i) an increase in the cell-substratum gap, (ii) a marked decrease in cell motility, (iii) reduced cell adhesion to glass.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts

Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double immunoelectron- microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and alpha- actinin with respect to these sites. (a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections. (b) FA sites were immunolabeled intracellularly for vinculin and alpha- actinin, with vinculin labeling situated closer to the membrane than alpha-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites. (c) CC sites were labeled intracellularly for alpha-actinin but not vinculin and were labeled extracellularly for fibronectin. (d) ECM sites were characterized by large separations (often greater than 100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both alpha-actinin and vinculin, the other for alpha-actinin alone. (e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and alpha-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.     

Adhesion of cells to protein carpets: do cells' feet have to be black?

In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cell-substratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein-covered substrata, their focal contacts may not always appear black in interference reflection microscopy.     

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.     

Ultrastructural localization of plasma membrane-associated urokinase- type plasminogen activator at focal contacts

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.     

Preliminary characterization of cell surface-extracellular matrix linkage complexes in cultured retinal pigmented epithelial cells

In this report, we describe the relative distribution of vinculin, talin, and fibronectin in cultured retinal pigmented epithelial cells from chick embryo eyes. We show that in these cells vinculin is present in both focal cell-substratum and cell-cell contacts, whereas talin is present only in the cell-substratum contacts. When cells are double-labeled for talin and fibronectin and viewed at the substratum level, fibronectin is not detectable and talin is concentrated in plaques corresponding to focal contacts. However, when the same cells are viewed at the apical level, both talin and fibronectin are present in a fibrillar pattern. In addition to fibrils which are both talin- and fibronectin-positive, there are areas which are either talin-positive and fibronectin-negative or, vice versa, talin-negative and fibronectin-positive. These observations indicate an interesting variability in the composition of transmembrane linkages in retinal pigmented epithelial cells in vitro.     

Cell matrix adhesions and localization of the vitronectin receptor in MCF-7 human mammary carcinoma cells

 Interference reflection microscopy (IRM) was used to evaluate the status of cell matrix adhesions in the MCF-7 human mammary carcinoma cell line. Focal contacts were concentrated at the periphery of individual cells or small cell clusters. Close contact was detected as a band at cell peripheries and as localized patches throughout the ventral face of cells. The MCF-7 cells also exhibited a distinctive reflection pattern of an intensity midway between that of either focal or close contact. This novel reflection pattern was located primarily at the periphery of cells and often obscured visualization of focal contacts in live cells. A similar distinctive pattern was absent from the normal tissue-derived MCF-10A mammary epithelial cell line. Immunofluorescence staining using an antiserum that cross-reacts with both α_vβ₃ and α_vβ₅ integrins revealed a distribution of the vitronectin receptor similar to that of the novel adhesion pattern as well as to that of focal contacts. In addition, IRM demonstrated the presence of ”tracks” associated with cells, which were also stained with the vitronectin receptor antiserum. The tracks are apparently residual material left behind as a result of cell migration. When MCF-7 cells were cultured in the absence of estradiol, the tracks were greatly diminished when visualized with either IRM or staining for the vitronectin receptor. In contrast, the addition of 17-β-estradiol to the medium resulted in an increased presence of the tracks as well as the development of extensive close contacts throughout the ventral surface of cells and cell clusters. Cells treated with the estrogen antagonist ICI 182,720 in the presence of estradiol had few associated tracks, indicating that the process leading to the formation of these structures is dependent on an estrogen receptor-activated pathway. However, the antagonist did not prevent the estradiol-induced formation of extensive close contacts. The extensive close contact as well as the increase in trailing material suggests that estradiol may promote breast tumor cell motility. However, this migratory activity may be mediated by both estrogen receptor-dependent and -independent pathways. Accepted: 29 May 1998     

Substrate-attached membranes of cultured cells isolation and characterization of ventral cell membranes and the associated cytoskeleton

We describe here an approach for the isolation and characterization of substrate-attached membranes of cultured cells. The procedure for ventral membrane preparation is based on a short incubation with ZnCl₂, followed by shearing with a stream of buffer. By varying the intensity of shearing it was possible to obtain reproducibly either entire ventral membranes or highly enriched focal contacts. The contacts with the substrate were retained in these preparations in an apparently intact state as determined by interference-reflection microscopy as well as by scanning and transmission electron microscopy. The formation of close contacts by the cells and by the isolated membranes was sensitive to changes of pH value. Thus in buffers at pH 7.0 to 7.2 the attachment was mediated predominantly by focal contacts, whereas at pH 6.0 the membranes reversibly formed extensive close contacts with substrate. The mechanical shearing removed most of the cytoskeleton, leaving attached only those components which were most tightly associated with the ventral membranes. Microtubules were easily removed, together with most of the intermediate filaments, whereas a considerable portion of the microfilament system was retained even after extensive shearing. Immunofluorescent labeling with antibodies to several microfilament-associated proteins, including actin, vinculin, α-actinin, filamin and tropomyosin, pointed to the specific interaction of each of these proteins with the isolated ventral membranes and focal contacts.     

Subcellular distribution of rhodamine-actin microinjected into living fibroblastic cells

The time course and pattern of incorporation of rhodamine-labeled actin microinjected into cultured fibroblastic cells were examined by fluorescence microscopy. Following microinjection, the fluorescent probe was incorporated rapidly into ruffling membranes, and within 5 min faintly fluorescent stress fibers were observed. Levels of fluorescence in ruffling membranes then tended to remain constant while fluorescence of the stress fibers continued to increase until approximately 20-min postinjection. Small, discrete regions of some microinjected cells displayed high levels of fluorescence that appeared initially approximately 5-10 min postinjection. I observed these small areas of intense fluorescence frequently near the cell periphery, which corresponded to focal contacts when examined with interference reflection optics. The results of this study show that a relationship exists between patterns of fluorescent actin incorporation in these cells and cellular areas or structures presumed to play a role in cell movement. These findings suggest that actin within stress fibers and the microfilament network of ruffling membranes undergoes a rapid turnover that may relate directly to the motility of the cell.     

Multiple labeling of cellular constituents by combining surface reflection interference and fluorescence microscopy

In this paper the technique for visualizing cytoskeleton in detergent-extracted cultured cells by surface reflection interference (SRI) microscopy after staining with the protein dye Coomassie Brilliant Blue (SRI-CooB technique) is used in conjunction with fluorescently-labeled antibodies or with other fluorescent probes to detect a number of constituents in the same cultured cell. Because SRI-CooB technique preferentially visualizes microfilament bundles along the ventral aspect of cells adhering to a glass substratum, we feel that this simple and rapid technique has great potential in studies of cell-substratum adhesiveness and of adhesion-related cytoskeletal organization.     

Orthogonal arrays of intramembrane particles in the supporting cells of the guinea-pig vestibular sensory epithelium

Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.     

Dynamic interactions of hepatocytes with fibronectin substrata: temporal and spatial changes in the distribution of adhesive contacts fibronectin receptors, and actin filaments.

We have studied the interactions, over several hours, of adult rat hepatocytes with fibronectin substrata in vitro by interference reflection microscopy and by laser scanning confocal immunomicroscopy using antisera against the fibronectin receptors, integrin alpha 5 beta 1 and nonintegrin Agp110, together with phalloidin, a specific marker for filamentous actin. Distinct alterations in the pattern of cell-substratum adhesive contacts, in the distribution of receptors, and in actin organization were observed with time in culture. Cells examined 2 to 3 h after inoculation had formed focal contacts at symmetrically distributed microextensions of the basal cell periphery that contained integrin alpha 5 beta 1, AGp110, and termini of actin filaments. On more prolonged incubation, over 5 to 6 h, increasing numbers of cells displayed a lamellar structure in close juxtaposition to the substratum circumscribing more than half of the basal cell surface and long filopodia emanating from the remaining part of the cell periphery. Integrin alpha 5 beta 1 and AGp110 were mainly concentrated at the inner and, to a lesser extent, outer boundaries of the lamellae, and actin filaments close to the basal surface overlayed the inner boundary of the adhesive lamella colocalizing with the receptors. Filopodial extensions contained neither receptor.     

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.     

Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum- bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti- adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.     

Ultrastructure of interstitial cells of Cajal at the gastro-oesophageal junction of the monkey (Macaca fascicularis)

The ultrastructure of the interstitial cells of Cajal (ICC) in the oesophagus of the monkey resembled that described in the oesophagus of other mammalian species but differed in their paucity and almost lack of smooth endoplasmic reticulum, caveolae and filaments. The plasmalemma of the ICC was in close contact (20- to 30-nm gaps) with that of smooth muscle cells. This may occasionally take the form of a desmosome, but gap junctions have not been observed. Vesiculated axon profiles, containing large granular or agranular vesicles were in close contact (20- to 30-nm gaps) with the plasmalemma of ICC. In a few vesiculated profiles a presynaptic density could be recognized. The intercalation of the ICC between the vesiculated axon profiles and the smooth muscle cells suggest a role in oesophageal motility. Between 3 and 21 days following bilateral vagotomy some ICC showed regressive changes such as increased electron density and shrinkage of the cytoplasm, crowding of the organelles and dissolution of the nuclear chromatin material. Axon profiles in the vicinity of the affected ICC contained glycogen granules suggesting injury. In late stages, the number of ICC and smooth muscle contacts was reduced. The results suggest that the vagus nerves exert a trophic influence on the ICC and that the intercellular relationships between ICC and smooth muscle cells possess a degree of plasticity. It is tentatively suggested that these vagal effects may be mediated via the oesophageal myenteric ganglia.