Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.

It is currently believed that the intermediate filaments of endothelial cells contain vimentin subunits exclusively. This inference, however, is derived from studies of only a few types of endothelial cells. By double indirect immunofluorescence and immunoelectron microscopy, we have now examined the endothelial cells of the micro- and macrovasculature of a variety of tissues and organs of adult chicken in vivo for their content of desmin and vimentin. Endothelial cells of the peritubular capillary in the renal cortex, the hepatic sinusoid, and the splenic sinusoid were found to contain only desmin; those of the exocrine pancreas capillary contained both desmin and vimentin; and the endothelial cells of the macrovasculatures and of all the other microvasculatures examined, including the vasa recta of the renal medulla, contained only vimentin. Such heterogeneity suggests that different types of adult chicken endothelial cells may have different embryological origins. To the extent that desmin and vimentin intermediate filaments may be functionally distinct, these results also suggest that different capillary endothelial cells may have different functional properties.

Monoclonal antibodies were generated to a purified preparation of the fascia adherens domains of the intercalated discs of chicken cardiac cell membranes. One of these antibodies, McAb 20, immunofluorescently labeled the Z lines of adult skeletal muscle, the Z lines and intercalated discs of adult cardiac muscle, and the dense bodies and dense plaques of adult gizzard smooth muscle. In addition, McAb 20 was found to label regenerating muscle cells in a cross-striated pattern much like that of Z lines in 24-h muscle cell cultures before the appearance of Z lines was detectable by phase or Nomarski optics and before the concentration of alpha-actinin occurred at the Z lines. Thus, McAb 20 appears to be directed against an antigen involved in early myofibrillar organization. Preliminary biochemical characterization of the antigen recognized by McAb 20 indicates that it is a high molecular weight doublet of over 5 X 10(5) kD that is highly susceptible to proteolysis. By virtue of its presence in Z lines, and its possible role in the end-on attachment of microfilaments to Z lines and membranes, we have named this protein zeugmatin (xi epsilon nu gamma mu alpha identical to yoking).

An integral membrane protein associated with sites of microfilament-membrane attachment has been identified by a newly developed IgG1 monoclonal antibody. This antibody, MAb 30B6, was derived from hybridoma fusion experiments using intact mitotic cells of chick embryo fibroblasts as the immunization vehicle as well as the screening probe for cell surface antigens. In immunofluorescent experiments with fixed cells, MAb 30B6 surface labeling is uniquely correlated with microfilament distributions in the cleavage furrow region of dividing chick embryo fibroblasts and cardiac myocytes in culture. The MAb 30B6 antigen in addition is associated with microfilament-membrane attachment sites in interphase fibroblasts at the dorsal surface, the adhesion plaque region at the ventral surface, and at junction-like regions of cell-cell contact. It is also found co-localized with the membrane-dense plaques of smooth muscle. The MAb 30B6 antigen is expressed in a wide number of chicken cell types (particularly smooth muscle cells, platelets, and endothelial cells), but not in erythrocytes. Some of the molecular characteristics of the MAb 30B6 antigen have been determined from immunoblotting, immunoaffinity chromatography, immunoprecipitation, cell extraction, and charge shift electrophoresis experiments. It is an integral sialoglycoprotein with an apparent molecular mass of 130 kD (reduced form)/107 kD (nonreduced form) in SDS PAGE. Another prominent glycoprotein species with an apparent molecular mass of 175 kD (reduced form)/165 kD (nonreduced form) in SDS PAGE is co-isolated on MAb 30B6 affinity columns, but appears to be antigenically distinct since it is not recognized by MAb 30B6 in immunoblotting or immunoprecipitation experiments. By virtue of its surface distributions relative to actin microfilaments and its integral protein character, we propose that the MAb 30B6 antigen is an excellent candidate for the function of directly or indirectly anchoring microfilaments to the membrane.

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.

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

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

Antibodies against chicken erythrocyte vimentin and gizzard desmin were affinity purified and then cross-absorbed with the heterologous antigen. They were used to study the in vivo distributions of these proteins in developing and mature myotubes by immunofluorescence microscopy of 0.5-2-micron frozen sections of iliotibialis muscle in 7-21-day chick embryos, neonatal and 1-d postnatal chicks, and adult chickens. The distributions of vimentin and desmin were coincidental throughout the development of myotubes, but the concentration of vimentin was gradually reduced as the myotubes matured and became largely undetectable at the time of hatching. The process of confining these proteins to the level of Z line from the initial uniform distribution occurred subsequent to the process of bringing myofibrils into lateral registry: in-register lateral association of several myofibrils was occasionally seen as early as in 7-11-d embryos, whereas the cross-striated immunofluorescence pattern of desmin and vimentin was only vaguely discerned in myotubes of 17-d embryos, just 4 d before hatching. In some myotubes of 21-d embryos, myofibrils were in lateral registry as precisely as in adult myofibers but desmin was still widely distributed around Z line in an irregular manner. Nevertheless, in many other myotubes of prenatal or neonatal chicks, desmin became confined to the level of Z line in a manner similar to that seen in adult myofibers, thus essentially completing its redistribution to the confined state of adult myofibers in coincidence with the time of hatching. In extracts from iliotibialis and posterior latissimus dorsi muscles of adult chickens, we detected a hitherto unidentified protein that was very similar to vimentin in molecular weight but did not react with our antivimentin antibody. We discuss the possibility that this protein was confused with vimentin in the past.

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.

We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the T-tubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system.

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.

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.

The ultrastructural localization of three cytoskeletal proteins, alpha-actinin, tropomyosin, and vinculin, in the brush border of epithelial cells of chicken small intestine and the smooth muscle cells of chicken gizzard was studied by immunofluorescence and immunonelectron microscope labeling of frozen sections of lightly fixed, intact tissues. In the immunoelectron microscope studies, a recently described new type of electron-dense antibody conjugate, imposil-antibody, has been successfully used, along with ferritin-antibody conjugates, in single and double immunolabeling experiments. In the intestinal brush border shows that vinvulin is sharply confined to the junctional complex close to the membrane region of the zonula adherens, in distinct contrast to the more diffuse distributions of the other two proteins. In the smooth muscle cells, the labeling patterns show that vinculin is sharply confined to the membrane-associated dense plaques, closer to the membrane than the alpha-Actinin is also present in the cytoplastic dense bodies, from which vinculin is absent. Tropomyosin is present diffusely distributed in the cytoplasm, but absent from both dense plaques and dense bodies. These findings with the muscle cells demonstrate, therefore, that the dense plaques and dense bodies are chemically and structurally distinct entities. The results with both tissues, along with those in previous papers (Geiger, 1979, Cell. 18:193-205.; Geiger et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:4127-4131), suggest that vinculin may play an important and widespread role in the linkage of actin-containing microfilament bundles to membranes.

Concanavalin A (Con A) was taken up to a limited extent by endocytosis in rabbit reticulocytes but not in rabbit erythrocytes. This process was observed by the use of ferritin-labeled Con A and transmission electron microscopy of thin sections of plastic-embedded cells. Furthermore, the extent of endocytosis among the reticulocytes decreased with the extent of their maturation, reticulocyte age being measured by ribosome configurations. These results are consistent with the proposal that there are domains in the membranes of reticulocytes in which the Con A receptors are laterally mobile, and can be clustered and endocytosed. These mobile domains exist, or are formed, within a larger framework of immobile membrane. During reticulocyte maturation, these domains are gradually eliminated, eventually disappearing upon formation of the mature erythrocyte. Possible molecular mechanisms for this proposed elimination process are discussed.

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

We have previously proposed that if the two half-layers of a membrane are different in their protein and lipid compositions, they may respond differently to some membrane perturbation (the bilayer couple hypothesis). This hypothesis has been applied to explain the changes in shape of human erythrocytes that are produced by a variety of amphipathic compounds. These compounds are presumed to intercalate by their hydrophobic ends into the lipid portions of the membrane; if the compounds are anions, the binding is preferentially to the outer half of the bilayer, if cations, to the inner half. It is proposed that such preferential binding causes an expansion of one half-layer relative to the other, with a corresponding change in cell shape. The predicted sidedness of these shape changes is now demonstrated in experiments with methochlorpromazine and 2,4,6-trinitrophenol. Under appropriate nonequilibrium or equilibrium or equilibrium conditions, both of these compounds are shown to be either crenators or cup-formers of the intact erythrocyte, depending upon which side of the membrane they are concentrated in. These results therefore strongly support the bilayer couple hypothesis.

We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.

The preparation, properties, and some applications of ferritin conjugates of two plant agglutinins, concanavalin A and Ricinus communis agglutinin, are reported. These conjugates serve as specific electron-dense stains for cell- and membrane-bound saccharide residues of the α- D -mannopyranosyl and ß- D -galactopyranosyl configurations, respectively, and as examples of a wide range of ferritin-plant agglutinin conjugates useful as high resolution saccharide stains. By using a technique for preparing flattened membrane specimens, it was found with a variety of mammalian cell plasma membranes (lymphocyte, lymphoma, and myeloma and normal, spontaneously and virally transformed fibroblasts) that the ferritin conjugates were localized exclusively to the exterior face of the membrane, with essentially none found on the cytoplasmic face. On the exterior face the topographical distribution of ferritin conjugates appeared to be random. The asymmetrical distribution of saccharide residues to the outer membrane face can be explained by an "assembly line" process whereby new plasma membrane is made from intracellular precursor membranes. It also suggests that the saccharide-containing components of the plasma membrane do not rotate at any appreciable rate from one membrane surface to the other.