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Steven C. Huhn
BA, Rutgers University, 2006
Thesis Advisor: Zhiyuan Shen, PhD
Monday, December 21, 2015
Mitosis is the process in which cells distribute newly duplicated chromosomes between two equivalent daughter cells. In higher eukaryotes, the process of chromosome segregation begins after nuclear envelope breakdown, when microtubules emanate from centrosomes and form a bipolar spindle array. The faithful execution of spindle establishment is critical for the precise distribution of chromosomes, and forces that disrupt faithful spindle formation are powerful impetuses for aneuploidy and genomic instability; key drivers of tumor establishment and evolution. BCCIP, a P21 and BRCA2 interacting gene, is an essential gene whose loss is tied to both genomic instability and tumor establishment. However, a complete picture of how BCCIP safeguards the genome is not well established. In Homo sapiens two major BCCIP isoforms that result from alternative splicing exist; designated BCCIPá and BCCIPâ. These two isoforms share an identical conserved domain spanning ~258 amino acids but are diversified by unique C-termini. The functional significance of this diversification is largely unknown. In mice and most other organisms, one isoform of BCCIP exists, which closely resembles BCCIPâ.
In our work we demonstrate that BCCIP, especially BCCIPá, is associated with both the interphase centrosome and the mitotic spindle poles. We demonstrate that BCCIP is primarily localized to the mother centriole where it functions in microtubule elongation and aids in the physical anchoring of microtubules to the centrosome. We show that the association of BCCIP with the centrosome is augmented by microtubules, requires dynein/dynactin activity, and demonstrate that BCCIP binds directly to tubulin through a central conserved domain common to both BCCIPá and BCCIPâ. Interestingly, we observe that in vivo, the ability to bind to centrosomes and the mitotic spindle is a feature of BCCIPá, but not BCCIPâ, while in mice and other eukaryotes, the sole BCCIPâ-like isoform is capable of assuming this function. In addition, the localization of BCCIPá to the spindle poles is concomitant with M-phase specific phosphorylation of BCCIPá, but not BCCIPâ, by the cytoskeletal regulator Src.
We observe that BCCIP loss results in a physical shrinking of the bipolar array spindle array, spindle pole broadening, and a loss of astral microtubules density. These defects contribute to errors in spindle orientation that induce a dramatic tilt of the spindle pole Z-axis in BCCIP deficient cells. Furthermore, we demonstrate that these faults result in the improper transduction of pulling forces from the spindle poles, concomitant with a decrease of the molecular tension between sister chromatids. In addition, we demonstrate that loss of BCCIP diminishes K-40 acetyl tubulin, a mark of stable microtubules. These aberrations contribute to a strong mitotic delay phenotype experienced by BCCIP deficient cells. Re-expression of BCCIPá, but not BCCIPâ, is capable of rescuing these defects in human cells, and the function of BCCIPá is partially dependent on M-phase specific phosphorylation by Src kinase.
In summary, we demonstrate BCCIP, especially BCCIPá, as a new component of the centrosome and mitotic spindle which regulates microtubule elongation and anchoring, enhances the stability of astral microtubules, and ensures proper spindle architecture. Furthermore, we establish a role for Src mediated cytoskeletal remodeling during mitosis. We demonstrate that the evolutionary divergence of BCCIPá and BCCIPâ in man may exist to fill a specific functional niche in cytoskeletal regulation. Altogether, we conclude that the tumor suppression function of BCCIP is partially mediated by stabilizing the minus end of the spindle apparatus and the faithful distribution of genetic material.