Fibronectin Receptor Internalization and AP-2 Complex Reorganization in Potassium-Depleted Fibroblasts

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.     

Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.     

Focal adhesion sites and the removal of substratum-bound fibronectin

Fibronectin was not removed from the substratum beneath focal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serum-containing medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibronectin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence of proteins in the incubation medium weakens the interaction of fibronectin with the substratum, thereby allowing the adsorbed protein to be removed from the substratum at sites of high stress. After removing fibronectin from the substratum, cells reorganized this material into patches and fibrils beneath cells, and the reorganized fibronectin colocalized with fibronectin receptors. Some of the patches of fibronectin were phagocytosed. The fibronectin fibrils were observed to be in register with actin filament bundles and sometimes translocated to the upper cell surfaces. It is proposed that removal of fibronectin from beneath focal adhesion sites is an example of how cells can modify their extracellular matrices through contractile activity.     

The coordinate organization of vinculin and of actin filaments during the early stages of fibroblast spreading on a substratum

Cultured normal fibroblasts adhere to their support essentially through the focal adhesion plaques which are greatly enriched with the 130 000 dalton protein, vinculin, along with the newly described 215 000 dalton protein, talin, and at which actin bundles terminate. In order to explore a role for vinculin in the formation of the adhesion plaques and of the actin bundles, we have studied and compared the development of these two cellular structures during the spreading of trypsinized and replated chicken embryonic fibroblasts. The techniques used were double indirect immunofluorescence and interference reflection microscopy. At the earliest stage of cell spreading observed, vinculin distributes into small patches that are located along actin filaments and at the basis of the ruffling membrane. At later spreading stage, vinculin markedly redistributes into larger striations which coincide with focal contacts. Some of these vinculin striations are associated with the ends of microfilaments while the others are not. These observations would suggest that two types of focal contacts can form simultaneously in early cell spreading. Hypotheses are made concerning the role of vinculin in the formation of the adhesive cell structures in the light of these new data and of previous reports on the subject.     

Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes

Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.     

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.     

The coordination between actin filaments and adhesion in mesenchymal migration

Mesenchymal cell motility is characterized by a polarized distribution of actin filaments, with a network of short branched actin filaments at the leading edge, and polymers of actin filaments arranged into distinct classes of actin stress fibres behind the leading edge. Importantly, the distinct actin filaments are characteristically associated with discrete adhesion structures and both the adhesions and the actin filaments are co-ordinately regulated during cell migration. While it has long been known that these macromolecular structures are intimately linked in cells, precisely how they are co-ordinately regulated is presently unknown. Live imaging data now suggests that the focal adhesions may act as sites of actin polymerization resulting in the generation of tension-bearing actin bundles of actin filaments (stress fibres). Moreover, a picture is emerging to suggest that the tropomyosin family of proteins that can determine actin filament dynamics may also play a key role in determining the transition between adhesion states. Molecules such as the tropomyosins are therefore tantalizing candidates to orchestrate the coordination of actin and adhesion dynamics during mesenchymal cell migration.     

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.     

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.     

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin–Hip1R interaction is involved in the cytoskeletal organization of coated pits.     

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.     

Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.     

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.     

α-Actinin: Immunofluorescent localization of a muscle structural protein in nonmuscle cells

Antibodies specific for the skeletal muscle structural protein α-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, α-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, α-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that α-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.     

Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts

To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane- lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the rate of internalization of receptor-bound transferrin and the residual membrane area of plasmalemmal clathrin-coated pits and vesicles were similarly decreased by approximately 90%. In contrast, the initial rate (< 5 min) of intracellular accumulation of the fluid- phase tracer HRP was not affected. Both in control and treated cells, the rate of HRP accumulation declined after approximately 5 min, and was twofold lower in treated cells, due to enhanced regurgitation. After correction for regurgitation, the endocytic rate constant was similar to measurements at shorter intervals and identical in control and treated cells. Similarly, the rate of internalization and the steady-state level of intracellular accumulation of two fluorescent lipid derivatives, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4- yl)amino]hexanoylglucosylsp hingosine (C6-NBD-GlcCer) and 1-[4- (trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were not affected by potassium depletion, indicating that the endocytic membrane traffic was equally preserved. Finally, the size distribution of primary endocytic particles that were accessible to HRP within 15 s before glutaraldehyde fixation was also indistinguishable in control and potassium-depleted cells. The simplest explanation is that clathrin polymerization is necessary to concentrate receptor-bound ligands in primary endocytic vesicles, but superfluous to the basic endocytic machinery in rat foetal fibroblasts.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

The effects of increased osmotic pressure on focal contacts and microfilament bundles of cultured fibroblasts

Incubation of normal substrate-spread mouse fibroblasts in hypertonic medium containing 0.25 M sorbitol leads first to partial shrinkage of cytoplasm, but later the cells restore the normal flattened cell shape. However, these sorbitol-incubated cells, in contrast to control cells, had no discrete bundles of microfilaments and considerably a diminished number of focal cell-substrate contacts. Thus, by incubation with sorbitol, distribution of actin microfilaments and morphology of cell-substrate contacts can be switched from one pattern to another without concomitant changes of the cell shapes.     

Calpain mediates integrin-induced signaling at a point upstream of Rho family members.

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that mu-calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of mu-calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of mu-calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac- and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.     

Functional role of Rho-kinase in ameloblast differentiation

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.     

A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy

The substratum adherent membrane of NRK cells in vitro has been studied using correlated interference reflection and surface replica electron microscopy. Structures visualized by interference reflection microscopy were identified in the subsequently prepared platinum-carbon replicas of the adherent membranes. Substratum adherent membranes were prepared using a hypotonic shock/cell shearing technique (lysis-squirting). Typically 20% of the original ventral membrane surface area and 50% of the original focal adhesion number were retained. Microfilament bundle termini, clathrin-coated sheets and pits, cytotic vesicles having a ridged surface and groups of membrane associated particles were well preserved in the replicas. Two types of isolated adhesion structures were found after lysis-squirting. In addition to typical elongate focal adhesions containing actin and vinculin, we report the existence of adherent membrane patches lacking microfilament bundles and negative for vinculin labelling, but coated with clathrin and identifiable in interference reflection microscopy as less dark than focal adhesions and having dot, U- or sinusoidal shapes.