Mitosis ensures the segregation of chromosomes to daughter cells by way of the mitotic spindle, which is composed of microtubules, microtubule associated factors, and the spindle matrix. This last component is able to be isolated from the other spindle components and is thought to be a supporting structure for the spindle. One prominent member of the spindle matrix found in vertebrates is lamin B, which causes defects in spindle morphology by some unknown mechanism. By using Xenopus egg extract, we can observe spindle formation in the absence of interphase effects from proteins like lamin B. In my studies, I have used spindles induced by Aurora A beads in egg extract to create three novel quantitative assays to study motor activity. I also characterized spindle formation in spindles induced by Aurora A beads and observed microtubule flux in this system. These spindles exhibited a unique dynamic behavior characterized by spindle elongation, which is likely supported by defects in the polar contribution to spindle flux. Regardless, these spindles showed predictable microtubule interactions in both parallel and antiparallel microtubule arrays based on observations from other spindle assembly systems. The motor activity assays derived from Aurora A beads were used to analyze the effect of lamin B on motor activity in this system. Lamin B was found to contribute to opposition of Eg5 forces in the spindle in all three assays. This observation was confirmed using spindles induced by sperm chromatin, where lamin B causes increased spindle length and multipolarity. Strangely, spindle flux was unaffected by lack of lamin B. This observation and evidence that lamin B interacts with dynein implied that it functions around the spindle to restrain microtubules through the use of motors which oppose Eg5 forces.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 73/05, Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry|
|Keywords:||Lamin, Microtubules, Mitosis, Spindle assembly|
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