Reconstitution of ciliary membranes containing tubulin.

Abstract

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.

Proteins of the Ciliary Axoneme Are Found on Cytoplasmic Membrane Vesicles during Growth of Cilia

“…Evidence of a direct association between tubulin and membrane, for example, has been reported [19–22]. It is possible that the vesicle association of RSPs reported here takes place between the membrane and IFT proteins, or occurs independently of membrane associated IFT proteins.…”

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron‐microscope radioautography after 3h‐fucose injection

“…It is conceivable that the drugs may bind directly to membranes. This has been shown to be the case for colchicine, and there is evidence that the plasma membrane of some cells may contain tubulin as a membrane component (Wolff and Bhattacharyya, 1975;Stephens, 1977Stephens, , 1983reviewed by Dustin, 1978). Thus, in the present work it is possible that the inhibition by colchicine and vinblastine of intracellular transport of membrane glycoproteins may be due to an action on microtubules, while the inhibition of exocytosis may be due to a direct action on components of the cell membrane involved.…”

Involvement of protein kinase C in 5‐HT‐stimulated ciliary activity in Helisoma trivolvis embryos

“…In addition to phosphatidylinositol metabolism, hydrolysis of phosphatidylcholine by phospholipase D (PLD) produces phosphatidic acid, which also leads to DAG formation through phosphatidic acid phosphomonoesterase activity (Nishizuka, 1992). In molluscan plasma membranes, the main phospholipids are phosphatidylcholine and phosphatidylethanolamine (Stephens, 1983), thus the action of PLD to produce DAG and subsequent activation of PKC is likely. In addition, findings from several systems suggest that phosphatidylinositol hydrolysis is not always required for activation of PKC.…”