Evidence that an RGD-dependent receptor mediates the binding of oligodendrocytes to a novel ligand in a glial-derived matrix.

A simple adhesion assay was used to measure the interaction between rat oligodendrocytes and various substrata, including a matrix secreted by glial cells. Oligodendrocytes bound to surfaces coated with fibronectin, vitronectin and a protein component of the glial matrix. The binding of cells to all of these substrates was inhibited by a synthetic peptide (GRGDSP) modeled after the cell-binding domain of fibronectin. The component of the glial matrix responsible for the oligodendrocyte interaction is a protein which is either secreted by the glial cells or removed from serum by products of these cultures; serum alone does not promote adhesion to the same extent as the glial-derived matrix. The interaction of cells with this glial-derived matrix requires divalent cations and is not mediated by several known RGD-containing extracellular proteins, including fibronectin, vitronectin, thrombospondin, type I and type IV collagen, and tenascin

Expression of a beta 1-related integrin by oligodendroglia in primary culture: evidence for a functional role in myelination.

We have investigated the expression of integrins by rat oligodendroglia grown in primary culture and the functional role of these proteins in myelinogenesis. Immunochemical analysis, using antibodies to a number of alpha and beta integrin subunits, revealed that oligodendrocytes express only one detectable integrin receptor complex (alpha OL beta OL). This complex is immunoprecipitated by a polyclonal anti-human beta 1 integrin subunit antibody. In contrast, astrocytes, the other major glial cell type in brain, express multiple integrins including alpha 1 beta 1, alpha 3 beta 1, and alpha 5 beta 1 complexes that are immunologically and electrophoretically indistinguishable from integrins expressed by rat fibroblasts. The beta subunit of the oligodendrocyte integrin (beta OL) and rat fibroblast beta 1 have different electrophoretic mobilities in SDS-PAGE. However, the two beta subunits appear to be highly related based on immunological cross-reactivity and one-dimensional peptide mapping. After removal of N-linked carbohydrate chains, beta OL and beta 1 comigrated in SDS-PAGE and peptide maps of the two deglycosylated subunits were identical, suggesting differential glycosylation of beta 1 and beta OL accounts entirely for their size differences. The oligodendrocyte alpha subunit, alpha OL, was not immunoprecipitated by antibodies against well characterized alpha chains which are known to associate with beta 1 (alpha 3, alpha 4, and alpha 5). However, an antibody to alpha 8, a more recently identified integrin subunit, did precipitate two integrin subunits with electrophoretic mobilities in SDS-PAGE identical to alpha OL and beta OL. Functional studies indicated that disruption of oligodendrocyte adhesion to a glial-derived matrix by an RGD-containing synthetic peptide resulted in a substantial decrease in the level of mRNAs for several myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and cyclic nucleotide phosphodiesterase (CNP). These results suggest that integrin-mediated adhesion of oligodendrocytes may trigger signal(s) that induce the expression of myelin genes and thus influence oligodendrocyte differentiation

Interaction of rat liver lysosomal membranes with actin.

Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt-washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane-concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free calcium concentration less than 10(-8) M). The actin cross-linking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10(-7) and 10(-9) M free calcium ions and inhibited by approximately 50% at concentrations of calcium greater than 0.5 x 10(-7) M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. The results suggest the existence of an integral lysosomal membrane actin-binding protein

Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis.

Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination

Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry.

The structure of rat liver vault ribonucleoprotein particles was examined using several different staining techniques in conjunction with EM and digestion with hydrolytic enzymes. Quantitative scanning transmission EM demonstrates that each vault particle has a total mass of 12.9 +/- 1 MD and contains two centers of mass, suggesting that each vault particle is a dimer. Freeze-etch reveals that each vault opens into delicate flower-like structures, in which eight rectangular petals are joined to a central ring, each by a thin hook. Vaults examined by negative stain and conventional transmission EM (CTEM) also reveal the flower-like structure. Trypsin treatment of vaults resulted exclusively in cleavage of the major vault protein (p104) and concurrently alters their structure as revealed by negative stain/CTEM, consistent with a localization of p104 to the flower petals. We propose a structural model that predicts the stoichiometry of vault proteins and RNA, defines vault dimer-monomer interactions, and describes two possible modes for unfolding of vaults into flowers. These highly dynamic structural variations are likely to play a role in vault function

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome