Analysis of microtubule assembly in vitro: I. Role of microtubule organizing centers (MTOCs) in controlling assembly of microtubules. II. Roles of the initiation proteins composing MTOCs and associated with brain microtubules in tubulin assembly.

Abstract

In the alga Polytomella basal body rootlets function as microtubule-organizing centers (MTOCs) controlling the positioning, initiation and patterning of cytoskeletal microtubules (Brown and Stearns, 1978). Procedures are described for the isolation and purification of intact basal body rootlet complexes. Quantitative electron microscopic and viscometry studies show that rootlet MTOCs are functionally intact and competent to initiate assembly of microtubules from both crude and phosphocellulose-purified brain tubulin preparations. Thin sections confirm that the microtubules are initiated from amorphous sites directly on rootlets in patterns closely resembling patterns of microtubules seen in situ. Biochemical analysis of the composition of rootlet-MTOCs shows that MTOCs consist of trypsin-sensitive protein(s) which can be extracted for purification by brief dialysis against a low ionic strength Tris-EDTA solution. Electron microscopic studies of treated complexes confirm that the complexes remain intact but incompetent to promote microtubule assembly. PAGE gels of the high speed supernatants of extracted complexes indicate a number of rootlet-associated proteins (RAPs) are removed. When RAPs are incubated at 37°C with phosphocellulose-purified tubulin in 0.1M PIPES, 1mM MgCl2, EGTA, pH 6.4 made 2mM in turbidometry studies show microtubules are assembled. Procedures are described for purification of the tubulin-initiating proteins of RAPs preparations and of microtubule-associated proteins (MAPs) obtained from temperature-cycled beef brain. Tubulin-initiating proteins (or TIPs) in RAPs preparations and microtubule-initiating protein ("the-MAP") in MAPs preparations are selected for by using centrifugation to pellet early assembly products (protofilaments) formed after brief incubation of fractions with tubulin. Phosphocellulose chromatography is used to further purify microtubule-initiating proteins in the protofilament pellets. PAGE gels of protein preparations show a single 330,000 mol. wt. protein ("the-MAP") in cycled brain tubulin (S-3) and 4 closely spaced 190-200,000 mol. wt. proteins in RAPs extracts (TIPs) function to promote assembly of protofilaments. The role of "the-MAP" and TIPs in promoting phosphocellulose-purified tubulin assembly is examined and compared using optical techniques (negative staining, turbidometry and viscometry). Negative staining studies show during initiation that "the-MAP" and TIPs stimulate formation of protofilaments and protofilament sheets as assembly intermediates which then form a complete microtubule wall. Intact rootlet-MTOCs are shown to initiate assembly of tubulin by the same mechanism. Turbidometry studies demonstrate that "The-MAP" and TIPs stimulate increases in initiation kinetics and suggest that "the-MAP" may also stimulate the elongation of microtubules. Viscometry studies show that "the-MAP" will stimulate microtubule elongation on rootlet-MTOCs whereas TIPs do not, but instead initiate new microtubules. Experiments using S35 labeled TIPs confirm that TIPs specifically function in initiation which is consistent with their composing MTOCs. Finally, electron microscopic studies of the structural properties of microtubules initiated by "the-MAP" and TIPs show the tubules have smooth surfaces. These microtubules become coated with arms and closely resemble S-3 microtubules if they are briefly incubated with crude MAPs, indicating that separate MAPs function to form arms on tubules. The microtubules are cold-sensitive and MAPs or "the-MAP" microtubules disassemble to form rings whereas TIPs microtubules disassemble to protofilaments. Analytical ultracentrifugation studies confirm these results. The main conclusion drawn from these studies is that microtubule-initiating proteins (TIPs) associate with MTOCs to control initiation of protofilaments during assembly of microtubules in vivo.