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.

Public Health Relevance

During the process of cell division, called mitosis, a critical step for maintaining healthy living cells is the segregation of the replicated chromosomes to the two daughter cells, which is accomplished by a dynamic macromolecular machine called the mitotic spindle. Errors in spindle function are associated the progression of cancer and other human disease. This proposal investigates how chromosomes are packaged during mitosis so that the spindle can physically separate them, how one particular protein called Clasp connects the microtubule fibers of the spindle to the chromosomes, and also aims to identify and characterize new proteins associated with microtubule fibers that also interact with membranes, another spindle component whose role in mitosis is poorly characterized.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM057839-12
Application #
8537923
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
1998-08-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
12
Fiscal Year
2013
Total Cost
$352,073
Indirect Cost
$120,473
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Heald, Rebecca; Cohen-Fix, Orna (2014) Morphology and function of membrane-bound organelles. Curr Opin Cell Biol 26:79-86
Schlaitz, Anne-Lore; Thompson, James; Wong, Catherine C L et al. (2013) REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture. Dev Cell 26:315-23
Wilbur, Jeremy D; Heald, Rebecca (2013) Cryptic no longer: arrays of CLASP1 TOG domains. Structure 21:869-70
Patel, Kieren; Nogales, Eva; Heald, Rebecca (2012) Multiple domains of human CLASP contribute to microtubule dynamics and organization in vitro and in Xenopus egg extracts. Cytoskeleton (Hoboken) 69:155-65
Fu, Hongxia; Freedman, Benjamin S; Lim, Chwee Teck et al. (2011) Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract. Chromosoma 120:245-54
Freedman, Benjamin S; Heald, Rebecca (2010) Functional comparison of H1 histones in Xenopus reveals isoform-specific regulation by Cdk1 and RanGTP. Curr Biol 20:1048-52
Walczak, Claire E; Heald, Rebecca (2008) Mechanisms of mitotic spindle assembly and function. Int Rev Cytol 265:111-58
Brown, Katherine S; Blower, Michael D; Maresca, Thomas J et al. (2007) Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle. J Cell Biol 176:765-70
Hannak, Eva; Heald, Rebecca (2006) Xorbit/CLASP links dynamic microtubules to chromosomes in the Xenopus meiotic spindle. J Cell Biol 172:19-25
Kenney, Renee Deehan; Heald, Rebecca (2006) Essential roles for cohesin in kinetochore and spindle function in Xenopus egg extracts. J Cell Sci 119:5057-66

Showing the most recent 10 out of 20 publications