title: Binding of exocytic vesicles from MDCK cells to microtubules in vitro.
abstract: Microtubules have been implicated in the transport of vesicles carrying newly synthesized proteins from the trans-Golgi network (TGN) to the cell surface. We have established a quantitative in vitro binding assay to investigate the putative interaction between these exocytic carrier vesicles and the microtubules at the molecular level. TGN-derived exocytic carrier vesicles, labeled with C6NBD-ceramide metabolites or viral glycoproteins, were obtained from polarized filter-grown MDCK II cells by perforation of the apical membrane with a nitrocellulose filter. These exocytic vesicles were incubated with taxol-polymerized tubulin and cytosol, layered on top of a 30% sucrose cushion and subjected to centrifugation. Quantitation of vesicles co-sedimenting with microtubules was done by measuring NBD-fluorescence of viral glycoproteins in the pellet and supernatant fractions. About 25% of the label sedimented through the cushion in the presence of microtubules and cytosol. Both apically and basolaterally targetted carrier vesicles containing influenza virus HA2 or vesicular stomatitis virus G protein, respectively, associated with the microtubules. Only 2-5% NBD-fluorescence was obtained in the pellet when no cytosol or microtubules were added to the vesicles. Negative-stain electron microscopy of resuspended pellets showed distinct microtubule-vesicle complexes. Heat inactivation or treatment of cytosol with N-ethylmaleimide (NEM), or trypsinization of vesicles inhibited the binding of vesicles to microtubules. Furthermore, coating of microtubules with brain microtubule-associated proteins abolished binding. These data suggest that NEM-sensitive cytosolic proteins are required for microtubule-vesicle association, and that the vesicles are bound via trypsin-sensitive receptor proteins on their surface.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
authors.forename: P
authors.initials: P
authors.lastname: Van der Sluijs
authors.forename: M K
authors.initials: MK
authors.lastname: Bennett
authors.forename: C
authors.initials: C
authors.lastname: Antony
authors.forename: K
authors.initials: K
authors.lastname: Simons
authors.forename: T E
authors.initials: TE
authors.lastname: Kreis
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D006056:Golgi Apparatus / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009711:Nucleotides / Q000378:metabolism; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D014357:Trypsin; D014404:Tubulin / Q000378:metabolism; D014709:Vero Cells
chemical_list: D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009711:Nucleotides; D014404:Tubulin; D014357:Trypsin; D005033:Ethylmaleimide