Studying Mitosis Using Xenopus Egg Extracts

University Of California Berkeley, Berkeley CA

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Abstract

DESCRIPTION (provided by applicant): Cell division is accompanied by dramatic changes in intracellular architecture as the mitotic spindle assembles, attaches to condensed chromosomes, and accurately distributes them to daughter cells. While spindle assembly has been studied for many years, the mechanisms governing it remain unclear, and we still do not know all of the factors involved. The general goal of the project is to elucidate the principles that underlie the dynamic events of mitosis, as well as to identify and study the roles of individual proteins. Since uncontrolled cell division is at the heart of the cancer problem, a molecular understanding of spindle assembly and function could lead to new approaches for cancer therapy. To study mechanisms of mitosis we use assays based on cytoplasmic extracts prepared from eggs of the frog Xenopus laevis that can recapitulate mitotic spindle assembly and chromosome condensation and segregation in vitro. The egg extract system has the advantage that cell cycle progression can be controlled and mitotic events induced under conditions that lack checkpoints. We have shown that magnetic beads coated with plasmid DNA induce spindle formation in extracts, illustrating the significant role that mitotic chromatin plays in this process. The Xenopus system is also well suited to investigate chromosome condensation, cohesion, and kinetochore formation and function, through the use of sperm chromosomes that can duplicate and undergo anaphase segregation in vitro. Our specific aims address fundamental questions with regard to chromosome architecture and spindle microtubule organization and dynamics. The general strategies take advantage of the open nature of the extracts, which allows temporally controlled and specific inactivation of individual components, biochemical identification of proteins and characterization of their activities and interactions, biophysical assays, and time-lapse fluorescence microscopy to study chromosome and microtubule morphogenesis at high resolution. Our aims are: (1) To study how linker histone H1 and its phosphorylation contribute to chromosome architecture and segregation. (2) To investigate the structure and function of Clasp, a (+TIP) protein crucial for chromosome- microtubule interactions during anaphase. (3) To identify and characterize microtubule- membrane linkers that function in cell division and morphogenesis.