Release from live choroid plexus of apical fragments and electrophoretic characterization of their synthetic products.

Protein synthesis and secretion by the choroid plexus (CP) has been implicated as a major source of certain proteins in cerebrospinal fluid (CSF), such as transthyretin. The suggestion that proteins are elaborated from CP through apocrine secretion has been borne out by the presence of newly labeled proteins in apical protrusions from CP (Agnew et al.: Cell and Tissue Research 208:261-281, 1980a). When the protrusions (aposomes) separate from the cells, they continue to incorporate labeled amino acids (Gudeman et al.: Tissue and Cell 19:101-109, 1987). In the present work the formation of aposomes in live CP explants indicated that these spheroids were not the result of fixation. Aposomes were also identified within rat CSF by immunohistochemistry with monoclonal directed against aposomes as well as with anti-transthyretin serum. The protein product of aposomes was characterized by 2-dimensional SDS-PAGE and compared to the protein products of whole CP tissue. Paradoxically, transthyretin, a heavily labeled protein in the tissue, was virtually undetected in the aposome synthetic profile. However, four other proteins were expressed in relatively equivalent amounts by the aposomes. The presence of mRNA in aposomes was detected with a poly dT probe, and the presence of actin was revealed by phalloidin staining of aposomes. These studies provide a more comprehensive definition of aposomes, but the functions of their secreted proteins remains to be determined.

Annulate lamellae: comparison of antigenic epitopes of annulate lamellae membranes with the nuclear envelope.

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.

Alpha-actinin and calmodulin interact with distinct sites on the arms of the clathrin trimer.

In the present study, the interaction of alpha-actinin and calmodulin with clathrin heavy chain are demonstrated using Western blot analysis and rotary shadowing electron microscopy. The results show that alpha-actinin and calmodulin bind the clathrin heavy chain. The interaction is specific and affected by calcium. However, the interaction of both proteins with the clathrin heavy chain is distinct; the proteins do not block each other's ability to bind, and they interact with different protein fragments of the clathrin heavy chain. Furthermore, using rotary shadowing the results show that alpha-actinin differentially affected the terminal region of the clathrin trimer. Whereas, the effects of calmodulin were most noticeably detected along the length of trimer arms. The possible existence of distinct binding sites on the arms of the clathrin trimer for these cytosolic proteins supports the contention that these cytosolic proteins play an important role in cellular trafficking.

Protein secretion by choroid plexus: isolated apical fragments synthesize protein in vitro.

Protein synthesis was studied in the isolated rat choroid plexus. When the choroid plexus was studied by transmission electron microscopy, membrane-bound structures were often observed in the ventricular space. These structures appear to bud from the apical surface of the epithelial cells. In the present study, we attempted to isolate these membrane-bound cellular fragments from the choroid plexus and to determine their ability to synthesize proteins. The apical fragments (aposomes) were isolated from the choroid plexus by allowing tissue explants to incubate in media (37 degrees C) for 1 h. The tissue was removed and the media, now containing aposomes, was incubated with [S35]methionine (100 microCi). The media was collected and analysed by SDS-PAGE followed by fluorography. Parallel [S35]methionine incubations were done with whole tissue explants. The SDS-PAGE protein derived from the aposomes was similar to the profile derived from the tissue. In addition, proteins detected in CSF had relative molecular weights comparable to the products synthesized by aposomes. These observations suggest that aposomes provide an additional route of entry for proteins into CSF.

The distribution of clathrin during amphibian oogenesis.

The distribution and content of clathrin during amphibian oogenesis was studied by indirect immunofluorescence and competitive ELISA assays. The antibody used for these studies was specific for the clathrin heavy chain and was capable of recognizing the antigen in a crude homogenate of amphibian oocytes. When this antibody was used to localize clathrin in fixed tissue slices of HCG-stimulated ovaries, differences were detected in the staining patterns of various sized oocytes. In cells less than 0.5 mm in diameter, the reaction appeared as a punctate pattern throughout the cytosol with a less intense reaction occurring in the region of the oolemma. As cell size increased, the intensity of the stain associated with the oolemma also increased, and the cytosolic reaction correspondingly decreased. To determine if this difference in intensity was due to an increase in clathrin concentration, competitive ELISA assays were carried out on extracts of variously sized oocytes. The results indicate that clathrin is preferentially synthesized in differently sized oocytes and that changes in the distribution and content of clathrin during oogenesis are related to developmental events occurring as the oocyte becomes progressively more specialized.

Paraphysectomy-induced stimulation of parathyroid glands in mature frogs (Rana catesbeiana): evidence for telencephalic regulation of parathyroid gland function.

The relationship of the paraphyseal-choroid plexus complex to parathyroid gland function was investigated in adult frogs. Light microscopy and morphometric analysis indicated that total parathyroid gland volume, cell volume and vascular volume doubled by 7-28 days after surgical removal of the paraphyseal-choroid plexus complex (paraphysectomy). This increase correlated with the appearance of large Golgi-associated vesicles, an increase in the apparent number of cytoplasmic dense-core granules, and PTH within the parenchymal cells as monitored by immunofluorescence. Twelve months after paraphysectomy, parathyroid glands became cystic with a central fluid-filled cavity surrounded by a stratified cuboidal cell layer. The parenchymal cells of cystic glands contained numerous cytoplasmic dense-core granules and were also positive for PTH. Radioimmunoassay of cystic parathyroid fluid indicated a PTH concentration of 2 micrograms/microliter; however, analysis by SDS-PAGE indicated a wide range of proteins in cystic fluid. The results of this study indicate that paraphysectomy induces stimulation of the parathyroid glands and suggest a role for the paraphyseal-choroid plexus complex in the regulation of amphibian parathyroid gland function.

Redistribution of clathrin heavy and light chains in anoxic pancreatic acinar cells.

In the present study, antibodies directed against clathrin light (33 kDa-36 kDa) and heavy (180 kDa) chains were used to confirm, by immunocytochemistry, that coated vesicles increase in number in the exocrine cells of pancreatic lobules incubated under anoxic (N2) conditions. The same antibodies were used to check whether or not the fibrillar aggregates, which appear under the same conditions on the cis side of the Golgi stacks of these cells, contain clathrins. By immunofluorescence, clathrin light chains were localized among zymogen granules and in small masses at the periphery of the Golgi complex in acinar cells incubated under N2. By electron microscopy, antibodies for light and heavy chains reacted with numerous coated vesicles located in clusters on the trans side of the Golgi stacks, scattered individually or in small clusters among zymogen granules throughout the apical region of the cell, and associated with both the apical and basolateral plasmalemma of the exocrine cells incubated under N2. The fibrillar aggregates cis to the Golgi complex stained less intensely and much less uniformly than the coats of the coated vesicle population. These findings suggest that the fibrillar aggregates which characteristically appear on the cis side of the Golgi stacks under anoxic conditions contain only small amounts of focally distributed clathrins. Their main components are probably other proteins whose relationship (if any) to clathrins and other clathrin cage constituents remains to be investigated. The findings also indicate that pancreatic acinar cells redistribute large amounts of clathrin, presumably from preexisting pools, when the transport of secretory and other proteins into the Golgi complex is inhibited. In control cells (incubated under O2-CO2), clathrin antigens had the same structural associations as in anoxic cells but the reactive sites were considerably less numerous and the reaction less intense.

The reorganization of the Golgi complex in anoxic pancreatic acinar cells.

An extensive reorganization of the Golgi complex (GC) was found in the acinar cells of pancreatic lobules incubated in vitro under conditions which inhibited ATP synthesis and thereby blocked the intracellular transport of secretory proteins. After 15-min incubation under N2 or in the presence of 1 mM dinitrophenol (DNP), transitional elements of the endoplasmic reticulum (ER) lost their protrusions, small peripheral Golgi vesicles decreased drastically in number, and fibrillar aggregates approximately 0.2 to 0.5 micron in diameter appeared on the cis side of the stacks of Golgi cisternae. These aggregates often contained vesicle-free, small (approximately 40 nm), globular cages and, occasionally, vesicle-free, clathrin-like cages. Fibrillar aggregates were also observed on the trans side of Golgi stacks. Other changes included the proliferation of GERL-elements ("rigid lamellae") and a striking increase in the population of coated vesicles trans to the Golgi complex and throughout the apical region of the anoxic acinar cells. All changes were found to be reversible provided the cells were incubated for less than 1 h under N2. These observations suggest that the fibrillar elements and the associated cages described in this study may play a role in the vesicular transport of newly synthesized proteins from the ER to the Golgi complex. Further work is needed to explore this possibility.

Evidence for the interaction of alpha-actinin and calmodulin with the clathrin heavy chain.

In the present study protein overlays were used to study the molecular interactions of clathrin with clathrin coat-associated proteins. Coated vesicles (CV) were isolated, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. The transfers were quenched and equilibrated in buffer, containing 1% Triton X-100. Alpha-Actinin and calmodulin, proteins known to interact with coated vesicles, were iodinated, placed in buffer, and incubated over the transfer for 1 h. After being rinsed extensively, the total amount of 125I associated with the filters was measured, and the filters were then processed for autoradiography. For alpha-actinin, the clathrin heavy chain and a series of lower molecular weight proteins were labeled. The binding of 125I alpha-actinin was inhibited with cold ligand and selectively released from the transfer with a buffer know to strip alpha-actinin from plasma membrane preparations. For 125I calmodulin the predominant binding site was also the clathrin heavy chain. Cold ligand inhibited binding and 60% of the detectable binding were calcium dependent. In addition, when these ligands were used in competition with each other, no significant inhibition was detected in the amount of binding associated with the clathrin heavy chain. These studies show that the clathrin heavy chain is a primary site of the clathrin cage receptive to intracellular interactions and furthermore suggest that the clathrin heavy chain consists of domains of biochemical specificity which may selectively affect the activities of coated vesicles.

Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells.

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 bran 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 effects of phospholipids on the properties of hepatic 5'-nucleotidase.

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.