Tissue expression of the vesicle protein pantophysin.

The cell-type restricted expression of cytoplasmic microvesicle membrane protein isoforms may be a consequence of the functional adaptation of these vesicles to the execution of specialized processes in cells of different specialization. To characterize the expression of the vesicle protein pantophysin, an isoform of the synaptic vesicle proteins synaptophysin and synaptoporin, we have prepared and characterized antibodies useful for the immunological detection of pantophysin in vitro and in situ. Using these reagents, we show by immunoblot analyses that pantophysin expression is not homogeneous but differs significantly between various bovine tissues. Furthermore, these differences are not exactly paralleled by the expression of other vesicle proteins of the SCAMP (secretory carrier-associated membrane protein) and VAMP (vesicle-associated membrane protein) types that have previously been localized to pantophysin vesicles in cultured cells. By immunofluorescence microscopy, pantophysin expression is seen predominantly in non-neuroendocrine cells with pronounced membrane traffic. Pantophysin staining codistributes with SCAMP and VAMP immunoreactivities in most instances but differs in some. Remarkably, pantophysin staining in liver is restricted to cells surrounding sinusoids and is not detectable in hepatocytes, similar to that of the SCAMP and VAMP isoforms as detected by our reagents in tissue sections.

Cytokeratins and cytokeratin filaments in subpopulations of cultured human and rodent cells of nonepithelial origin: modes and patterns of formation.

Using immunofluorescence microscopy, we observed that in several established cell culture lines derived from different nonepithelial tissues and species, cells spontaneously emerge, usually at low frequencies, which contain cytoplasmic structures decorated by antibodies specific for cytokeratins 8 and 18. This phenomenon was further examined at both the protein (gel electrophoreses of cytoskeletal proteins, followed by immunoblotting) and the RNA (Northern blots, "nuclear run-on" analysis, in situ hybridization) level. Positive cell lines included simian virus (SV40)-transformed human fibroblasts (HF-SV80, WI-38 VA13), human astrocytic glioma cells (U333 CG/343MG), rat (RVF-SMC) and hamster (BHK-21/13) cells derived from vascular smooth muscle and murine sarcoma MS-180 cells. In two cell lines (HF-SV80 and BHK-21/13), the frequency of the cytokeratin-containing cells and of the cytokeratin fibril arrays per cell was drastically increased upon treatment with 5-azacytidine. The structural appearance of the cytokeratins was variable in the different cell lines but could also differ among cells of the same culture: While small granular or comma-shaped structures or bizarrely shaped filament arrays prevailed in WI-38, RVF and normally grown BHK-21 cells, most of the other lines revealed extended normal-looking, fibrillar arrays. In one line (MS-180), the appearance of cytokeratins was associated with a morphological change, as it was only found in a subpopulation of cells that had lost their typical elongated and spindle-shaped phenotype and assumed a rounded ("coccoid") shape. Our results show that the expression of the genes encoding cytokeratins 8 and 18 is not necessarily restricted to programs of epithelial differentiation and that factors stochastically effective appear in cultured cell lines that allow the synthesis of these cytoskeletal components. Mechanisms possibly involved in this spontaneous and selective advent of cytokeratins 8 and 18 and implications for tumor diagnosis are discussed.

Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules.

During adipose conversion of murine 3T3-L1 cells, the arrangement of vimentin intermediate filaments (IFs) changes from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules. The fully developed cage complex surrounding the lipid globule consists of a monolayer of groups of regularly spaced vimentin IFs that in turn is closely ensheathed by a special endoplasmic reticulum cisterna. The same IF cage is also seen in other adipocytes in culture and in tissues. The specificity of the association of lipid globules with vimentin IFs during adipose conversion is discussed as a special form of compartmentalization supporting adipogenesis and is taken as an example of a possible IF function in relation to a cell differentiation process.