Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies.

Abstract

Microtubules were isolated from unfertilized eggs of the sea urchin with the use of the anti-tumor drug taxol. In addition to tubulin, prominent high molecular weight (Mr;205,60 microtubule-associated proteins (MAPs) were identified as well as MAP species of Mrs 77,000, 100,000, and 120,000. The microtubules were covered with both short periodic arms and longer filamentous arms,-both classes of which appeared to.crosslink the microtubules into bundles; Monoclonal antibodies were prepared to an unfractionated MAPs preparation. We isolated clonal hybridoma' lines producing antibodies.to tubulin and totfour nontubulin proteins of Mrs 235,000, 205,000, 150,000, and 37,000. All antibodies strongly and specifically stained the mitotic spindle of dividing sea urchin eggs. 1.0 mM. The cytosolic extract was warmed to 370C for 5 min to assemble microtubules, chilled on ice for 15 min, and centrifuged at 22,500 x g for 30 min at 2VC through a cushion of 10% sucrose in lysis buffer containing 20 ,uM.taxol and 1.0 mM GTP. As described previously (11), the microtubule pellet was resuspended (total volume, 1/4 to 1/5 the volume of the cytosolic extract solution) and washed in 0.1 M Pipes, pH 6.6/1 mM EGTA/1 mM MgSO4 (PEM buffer) containing 1 mM GTP Abbreviation: MAP, microtubule-associated protein.6259

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.

“…These proteins are thought to modulate the assembly and stability of microtubules and to mediate the interaction of microtubules with other cellular components. A number of proteins have been shown to coassemble with microtubules in vitro (reviewed by Olmsted, 1986) and associate with interphase and spindle microtubules (Vallee and Bloom, 1983;Bloom et al, 1984). Since most of the studies of microtubule-associated proteins (MAPs) ~ have been conducted in systems not amenable to genetic analysis, the in vivo functions of these MAPs are still uncertain.…”

Interacting genes that affect microtubule function: the nc2 allele of the haywire locus fails to complement mutations in the testis-specific beta-tubulin gene of Drosophila.

“…MAPs participate in microtubule functions such as transport of organelles along microtubule tracks (Vale et al 1985) and the movement of cells by flagella and cilia (Warner and Mitchell 1980;reviewed in Gibbons 1981). MAPs are also likely to be important in the movement of chromosomes by mitotic and meiotic spindles (Izant et al 1982;Vallee and Bloom 1983). Some MAPs have been found in association with other cellular components such as intermediate filaments and microfilaments, where they could be involved in linking microtubules to other structures in the cell (Schliwa and van Blerkom 1981;Griffith and Pollard 1982;Leterrier et al 1982).…”

Microtubule-mediated Src Tyrosine Kinase Trafficking in Neuronal Growth Cones

“…With regular fixation, the percentage of Src2/MT overlap was only slightly higher in the total Src2 signal. (K-M) Biochemical cell fractionation of Aplysia CNS tissue to enrich for MTs after a modified protocol originally developed by R. Vallee and G. Bloom (Vallee, 1982;Vallee and Bloom, 1983) (see Materials and Methods for details). (K) SDS-PAGE (10%) and silver staining of homogenate (H), high-spin pellets (P1, P2, and P3), and supernatant (S1, S2 and S3) fractions.…”