MECHANISMS OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

该项目的总体目标是确定分子和 调节微管组装和移动结构机制 染色体 我们的方法仍然是发展一个孤立的 从海胆卵制备有丝分裂纺锤体， 分析主轴的结构、组成和功能。 我们也 现在集中在钙调节纺锤体组装的假设上， 在体内起作用，有丝分裂器内源性膜 局部控制刺内钙离子（CA++）浓度。 我们有 开发了一种使用EGTA裂解缓冲液进行分离的技术， 储存对微摩尔CA++不稳定的无膜有丝分裂纺锤体。 对于EGTA分离的纺锤体，我们打算继续分析蛋白质 组成，超微结构，微管极性，以及 其中钙使微管解聚并导致微管缩短， 纺锤体纤维 我们正在研究最佳缓冲条件， 重新激活生命般的组装-拆卸特性， EGTA分离的纺锤体，并将分离的卵微管蛋白用于 重组缓冲区。 荧光标记的微管蛋白 二氯三嗪基荧光素（DTAF）将帮助我们定位的网站， 微管蛋白的掺入和从微管的解离 EGTA隔离主轴。 我们将调查的纳入和流量 微管蛋白在活纺锤体中的作用 细胞 我们还打算分离保留有丝分裂的有丝分裂器。 正常结构的刺内膜，我们将研究他们的 超微结构和蛋白质组成（比较它们与无膜 EGTA分离的纺锤体），以及它们隔离CA++的能力。 的 膜在调节刺内CA++浓度中的作用将 通过测量细胞内CA +波动进行体内研究， CA++选择性微电极，通过监测金霉素的变化 荧光或水母发光蛋白发光。 我们的观察将与 各种显微技术（偏振、暗场、荧光等） 和图像增强视频延时记录系统。 最终我们 希望获得一种分离有丝分裂模型系统， 缓冲条件，将使我们能够重新激活生命般的染色体 动作

The overll objective of this project is to determine the molecular and structural mechanisms which regulate microtubule assembly and move chromosomes. Our approach continues to be the development of an isolated mitotic spindle preparation from sea urchin eggs which permits direct analysis of spindle structure, composition, and function. We are also focusing now on the hypothesis that calcium regulates spindle assembly and function in vivo, and that membranes endogenous to the mitotic apparatus locally control intraspindle calcium ion (CA++) concentrations. We have developed a technique which uses an EGTA lysis buffer for isolating and storing membrane-free mitotic spindles that are labile to micromolar CA++. For the EGTA-isolated spindles we intend to continue analyzing the protein composition, ultrastructure, microtubule polarity, and the mechanism by which calcium depolymerizes the microtubules and causes shortening of the spindle fibers. We are investigating optimum buffer conditions for reactivating life-like assembly-disassembly characteristics in the EGTA-isolated spindles and will isolate egg tubulin to use in the reassembly buffers. Tubulin that has been fluorescently labelled with dichlorotriazinyl fluorescein (DTAF) will help us to localize sites of tubulin incorporation and dissociation from the microtubules of EGTA-isolated spindles. We will investigate the incorporation and flux of tubulin in living spindles by microinjecting DTAF-tubulin into mitotic cells. We also intend to isolate mitotic apparatuses that retain the normal structure of the intraspindle membranes, and we will study their ultrastructure and protein composition (comparing them to the membrane-free EGTA-isolated spindles), as well as their ability to sequester CA++. The role of the membranes in regulating intraspindle CA++ concentrations will be investigated in vivo by measuring intracellular CA + fluctuations by CA++ selective microelectrodes, by monitoring changes in chlorotetracycline fluorescence, or aequorin luminescence. Our observations will be made with various microscopy techniques (polarization, darkfield, fluorescence, etc.) and an image-intensified video time-lapse recording system. Ultimately, we hope to achieve an isolated mitotic model system which, with appropriate buffer conditions, will enable us to reactivate life-like chromosome movements.