ASSEMBLY AND BUNDLING OF MARGINAL BAND MICROTUBULE PROTEIN - ROLE OF TAU

Microtubule protein extracted from dogfish erythrocyte cytoskeletons b y disassembly of marginal bands at low temperature formed linear micro tubule (MT) bundles upon reassembly at 22 degrees C. The bundles, whic h were readily visible by video-enhanced phase contrast or DIC microsc opy, increased in length and thickness with time. At steady state afte r 1 hour, most bundles were 6-11 mu m in length and 2-5 MTs in thickne ss. No inter-MT cross-bridges were visible by negative staining. The b undles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-P AGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high m olecular weight MAPs such as MAP-2 or syncolin. In addition, late frac tions of MT protein obtained by gel filtration were devoid of high mol ecular weight proteins but still produced MT bundles. The marginal ban d tau was salt-extractable and heat-stable, bound antibodies to mammal ian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the p resence 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 acti vity, and salt-induced unbundling was reversible with desalting. Howev er, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of margi nal 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. (C) 1994 Wiley-Liss, In c.