The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles . When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinitypurified anticlathrin IgGs-one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. (Louvard,
Abstract. Annulate lamellae (AL) are a membranous structure frequently observed in differentiating gametes and tumor cells. In spite of numerous morphological studies, the function and biochemical composition of this membrane system are not well understood. In this study, we have examined the AL membrane system of vinblastine-treated mouse L cells using immunocytochemistry and Western blot analysis. Our results show that antibodies directed against nuclear envelope lamins, i.e., lamins A, B, and C, did not cross react with constituents of the AL membrane system. Furthermore an AL-specific antibody failed to react with the nuclear envelope and reacted minimally producing only a background stain over other cellular components. The data suggest that the AL membrane system has a distinct molecular make-up that is antigenically distinct from that of other subcellular structures.NULATE lamellae (AL) ~ are a network of intracellular membranes that have been described in numerous studies over the past 30 years (11). These membranes are often observed in rapidly dividing cells, germ cells, and cells at the onset of differentiation (11,12,18,19). However, the function served by this membrane system has yet to be elucidated. This organelle consists of a parallel stack of membranous cisternae trimmed with dense staining fibrillar plaques. Characteristically, the membranes contain pores. These intramembranous pores are morphologically similar to those found in the nuclear envelope. This has led to the speculation that these two membrane systems are structurally and perhaps functionally related (18).In this study we have compared the antigenicity of the nuclear envelope and the AL membrane system with the hope of identifying epitopes common to both membrane systems. As a marker for the nuclear envelope we have used antibodies directed against the nuclear lamins. In recent years the nuclear lamins have been shown to be a highly conserved family of proteins associated with the inner aspect of the nuclear envelope (6, 15). Lamins A, B, and C have been purified and extensively characterized (5, 7). Each is believed to form part of a protein complex that structurally supports the envelope during mitosis (5). An immunocytochemical approach was used to determine whether the lamins are present in the AL membrane system. In addition an antibody specific for the AL membrane system was detected in a serum sample taken from a patient having lupus erythematosus. This AL specific antibody was used to determine the cellular distribution of AL antigens. Abbreviations usedin thispaper:AL, annulate lamellae; TEM, transmission electron microscopy. Materials and Methods Reagents Induction of AL MembranesMouse L cells were grown in RPMI 1640 supplemented with 10% FCS and 1% penicillin-streptomycin. Under standard conditions the cells were cultured to near-confluency. Afterwards, the cells were cultured in medium containing vinblastine sulfate (0.1 pg/ml) for 36 h. The cell cultures were then processed for either transmission electron microscopy ...
Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated Staphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent . The specificity of the immunoadsorption was monitored by SDS PAGE analysis and by competitive ELISA assays . SDS PAGE of the material immunoadsorbed from a fraction of porcine brain smooth microsomes showed a selective enrichment in a 180,000 mol wt protein. In an ELISA assay, this protein competed effectively-in binding anticlathrin-with clathrin extracted from a coated vesicle preparation . When the immunoadsorbed fraction was examined by electron microscopy, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles . Upon extensive dialysis (against MES buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin .This study demonstrates that anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles . In addition, the ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution .The ubiquitous presence of coated vesicles in eukaryotic cells has generated a great deal of interest and speculation concerning the role(s) these organelles play in cellular processes . Recent studies have shown that coated vesicles are involved in receptor-mediated endocytosis (2,4,14,15,17,38,40,43), formation of primary lysosomes (13), membrane recycling (5, 18, 19), and intracellular vesicular transport (11-13, 36, 41).The pertinent evidence has been derived either from morphological observations of cells actively engaged in one of the processes mentioned, or from cell fractionation studies . Both of these general approaches have technical limitations when applied to the study of coated vesicles because of the transient existence of these structures and the fragility of their clathrin coat. Besides, with these procedures, many important questions concerning the mechanisms involved in the assembly and/or disassembly of coated vesicles in situ, or the fate of the clathrin cage upon disassembly from the vesicle are virtually impossible to answer .In an attempt to improve on available isolation procedures and to solve some pending problems concerning the formation and function ofcoated vesicles, we have developed a technique that uses anticlathrin antibody for the affinity separation of coated vesicles from crude fractions of tissue homogenates . In this report, we show that formaldehyde-fixed, heat-inactivated Staphylococcus aureus cells can be used as a solid phase immunoadsorbent for the isolation of coated vesicles. With this procedure, it is possible to: (a) increase the ef...
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