Amitahba Gupta

Interdisciplinary Training

Amitahba Gupta

"Elucidating the multiple functions of CLASP in kinetochore biorientation"

The accurate segregation of chromosomes is essential to prevent aneuploidy, a hallmark of cancer. One
key step in this process is the stable attachment of sister chromatids to microtubules emanating from
opposite spindle poles. These attachments are mediated by kinetochores, large proteinaceous
structures that assemble on chromosomes. The mechanism of attachment is an intricate process - the
kinetochores first bind to the side of the microtubule lattice and then bind to the microtubule ends
where they endure a large amount of force as the microtubules shrink and grow. Additionally, inhibitory
Aurora B-phosphorylation of some kinetochore components must be removed by the protein
phosphatase, PP1, before kinetochore-microtubule attachment can occur. These steps have previously
been studied in vitro using purified kinetochores containing ?core? components. However, since these
purified kinetochores lack certain components that could significantly affect these steps, our
understanding of this vital process remains incomplete. One such component is the microtubule-
associated protein, CLASP, which has been shown to be important for attachment. I therefore, aim to
recreate kinetochores containing CLASP to better understand the in vivo mechanism of kinetochore-
microtubule attachment. Using pioneering biochemical techniques from the Biggins laboratory, I will be
able to recreate these CLASP-containing kinetochores and ascertain the effect on kinetochore
composition and dephosphorylation of Aurora B targets. I will then utilize the biophysical techniques
developed in the Asbury laboratory to better understand which step of kinetochore-microtubule
attachment is affected by CLASP. In this way, I will expand our current understanding of how
chromosome segregation occurs.