The proposed research investigates the mechanisms by which microtubule assembly is regulated during the cell in eggs of the African frog Xenopus laevis. The approach involves the investigation of factors which affect microtubule assembly in vitro, and which might regulate assembly in vivo. Two such factors have been identified. XMAP, a factor which promotes microtubule assembly, has been purified 1000-fold from eggs. XMAP specifically promotes elongation of the microtubule plus- ends by altering both the rate of tublin loss and addition. XMAP is an M-phase phosphorprotein, suggesting that phosphorylation regulates XMAP function. The second factor, found in Xenopus oocytes, specifically inhibits elongation of microtubule plus-ends. The proposed experiments address the following questions regarding these factors: (1) Does XMAP bind to microtubules in vitro and in vivo? Immunofluorescence and electron microscopy will be used to visualize the binding of XMAP to microtubules both in vitro and in the intact egg. Attention will be paid to the localizaton of XMAP to specific classes of microtubules. (2) How does XMAP promote assembly at the plus-end without affecting the minus-end, and how does its end specificity affect free microtubules? The effect of XMAP on the rates of tubulin addition and loss at the minus-end will be directly measured, as will the effect of XMAP on microtubule nucleation and microtubule dynamics at steady state. (3) Does phosphorylation regulate XMAP function, and thereby regulate microtubule assembly? Phosphorylated and dephosphorylated XMAP will be assayed for promotion of tubulin assembly in vitro to determine whether phosphorylation affects XMAP activity. (4) What protein or factor is responsible for inhibition of microtubule assembly in the oocyte? The inhibitor will be isolated, and gel-filtration or immunoprecipitation will be used to determine if it binds tubulin monomer. (5) How is the inhibitor inactivated during meiosis? Metabolic labeling of oocytes with 32PO4 of the 35S-methionine and immunoprecipitation will be used to study inactivation of the inhibitor by degradation or post-translational modification. (6) Are proteins related to XMAP or the inhibitor found in other cell types, or in other organisms? A functional assay will be used to screen other cell types for the characteristic plus-end effects of these factors on microtubule assembly. In addition, specific antisera will be used to screen for related proteins by immunofluorescence and immunoblotting.
|Schroeder, M M; Gard, D L (1992) Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs. Development 114:699-709|