Controlled extraction of intact gill tissue, isolated cilia or reconstituted membrane vesicles with Nonidet P-40 at greater than 4 times the critical micelle concentration, or with octyl glucoside at the critical micelle concentration, delipidates the membrane, leaving a membrane remnant or skeleton of membrane tubulin and associated proteins. This skeleton consists of a disordered reticular protein network in reconstituted membrane vesicles and a similar but more compact sleeve in cilia of extracted tissue. The membrane skeleton is closely apposed to the axoneme and is attached to the outer doublets by fine radial bridges having a 20–24 nm longitudinal periodicity, supporting earlier observations made utilizing a lipophilic cross-linking agent. Higher concentrations of detergent solubilize the membrane tubulin-protein complex, producing 5–10 nm particulate material of low sedimentation coefficient. Dilution of an octyl glucoside solution to below the critical micelle concentration results in disappearance of the particles and reformation of the membrane, indicating that the particles are protein-detergent micelles and not denatured protein. Freeze-fracture electron microscopy reveals no comparable-sized natural particles in the ciliary membrane proper. The reticular material of the membrane skeleton contains tubulin, demonstrated on Lowicryl K4M thin sections by a rabbit polyclonal antibody to sea-urchin egg cytoplasmic tubulin, using gold-labelled secondary antibody. Minimal cross-reactivity is detected prior to Triton-delipidation, suggesting that most membrane tubulin antigenic sites are buried within the bilayer and that the tubulin is not simply adsorbed to the lipid bilayer.
Evidence that tubulin forms an integral membrane skeleton in molluscan gill cilia
R.E. Stephens, S. Oleszko-Szuts, M.J. Good; Evidence that tubulin forms an integral membrane skeleton in molluscan gill cilia. J Cell Sci 1 November 1987; 88 (4): 527–535. doi: https://doi.org/10.1242/jcs.88.4.527
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