Assembly and bundling of marginal band microtubule protein: role of tau.
Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22 degrees C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 microns in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were "unbundled" by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor.
Sanchez
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Department of Biological Sciences, Hunter College of CUNY, New York, New York.
Cohen
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D000818:Animals; D002850:Chromatography, Gel; D004284:Dogfish / Q000097:blood; D004912:Erythrocytes / Q000737:chemistry / Q000648:ultrastructure; D006358:Hot Temperature; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009024:Morphogenesis; D017239:Paclitaxel / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D016875:tau Proteins / Q000502:physiology*
D046911:Macromolecular Substances; D008868:Microtubule Proteins; D016875:tau Proteins; D012965:Sodium Chloride; D017239:Paclitaxel