Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule - associated proteins with monoclonal antibodies ( hybridoma / immunofluorescence microscopy / immunoblot / fertilization / development ) RICHARD

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;205,000350,60 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. All four of the immunoreactive, nontubulin species behaved as MAPs during microtubule isolation. Thus, we have identified a variety of sea urchin MAPs by biochemical, ultrastructural, and ixnmunochemical means. The immunochemical experiments demonstrated that four of these proteins are microtubule-assoeiated components of the mitotic. spindle. We suggest that those proteins that we observed as cross-bridges between the isolated microtubules may be either. structural or functional components of the spindle. Cytoplasmic microtubules are involved in a number of fundamental processes in eukaryotic cells. Although the question of how microtubules assemble and become organized into the variety of arrangements. that-have been observed in cells has been the subject of much recent work, little is known about how microtubules perform their cellular functions. The most basic role for microtubules is in cell division. Microtubules have long been known to be the predominant structural components of the mitotic spindle (1). Nevertheless, their precise role in the mechanism of mitosis is still poorly understood. Sea urchin eggs provide an attractive experimental system for the study of mitosis. Homogeneous populations of synchronously dividing eggs can be obtained readily in large amounts sufficient for biochemical analysis. Mitosis may. be monitored readily and the mitotic spindles may be manipulated experimentally in vivo. The primary function of microtubules in this system is in mitosis (see ref. 2); thus, the biochemical analysis of sea urchin egg microtubules should provide a relatively direct route for the molecular analysis of the mitotic spindle. Several studies that have appeared have sought to characterize the protein components of sea urchin egg microtubules in vitro. Kane (3) found that microtubules would not self-assemble readily from extracts of sea urchin eggs under conditions that were used for the purification of vertebrate brain microtubules. Kuriyama (4) succeeded in assembling and purifying microtubules from sea urchineggs after chromatographic fracThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S.C. §1734 .solely to indicate this fact. tionation of the egg cytosolic extract. The purified microtubules -contained only tubulin, the major component of microtubules. Kellerand Rebhun (5) succeeded in assembling and purifying microtubules, using sea urchin mitotic spindles as starting material. lu addition to tubulin, the purified microtubules contained a protein of Mr 80,000. This protein potenttially vrepresented a microtubule-associated protein (MAP), ;,though no other proteins similar to those identified in mammalian brain tissue or cultured cells (for review, see ref. 6) were -observed. In addition, the microtubules were smooth-walled, Jacking the fine filamentous arms characteristic of the high molecular weight mammalian brain proteins MAP 2 (7, 8) or MAP 1 (9) or the heavier arms characteristic of ciliary or flagellar dynein (10). We sought to identify proteins associated with sea urchin microtubules with the ultimate goal of identifying microtubuleassociated components of the mitotic spindle. We report here on the isolation of MAP-containing microtubules from sea urchin eggs, using a procedure (11) that takes advantage of the microtubule assembly-promoting activity of the anti-tumor drug taxol. The microtubules are covered with fine arms that resem.ble similar features observed in sea urchin mitotic spindles. We raised monoclonal antibodies to the MAPs and used these antibodies to demonstrate directly the presence of four distinct MAPs in the mitotic spindle. MATERIALS AND METHODS Preparation of Microtubules and MAPs. Microtubules and MAPs were prepared by using a modification of the taxol-dependent procedure described previously (11). Unfertilized sea urchin eggs were dejellied by passage through a Nitex screen and were washed three times with >5 vol of lysis buffer (0.1 M Pipes, pH 6.6/5 mM EGTA/1 mM MgSO4/0.9 M glycerol or 0.5 M mannitol/2 mM phenylnethylsulfonyl fluoride/i mM dithiothreitol/100 ftg of soybean trypsin inhibitor per ml). The eggs were resuspended to 3 vol in lysis buffer, homogenized in a Dounce tissue grinder, and.centrifuged at 30,000 X g for 30 min at 20C. The supernate was recovered and centrifuged at 135,000 x g for 90 min at 20C. Taxol was added to this second supernate (cytosolic extract) to 20 ,uM, and GTP was added to 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.