Dr. Sue Biggins

Regulation of chromosome segregation.

How do cells faithfully inherit a complete set of chromosomes during every cell division? My laboratory is focused on elucidating the mechanisms that govern chromosome segregation. Because aneuploidy is a hallmark of all cancers and many birth defects, studies on chromosome segregation are critical to understanding how cells maintain genomic stability and prevent disease. Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA sequences and attach to spindle microtubules. Sister kinetochores contain multiple microtubule binding sites that must all make bioriented attachments to microtubules from opposite poles. Once proper bioriented attachments are made, the microtubule pulling forces generate physical tension on the sister chromatids. Defects in assembling bioriented kinetochore attachments are detected by the spindle checkpoint that halts the cell cycle until the errors are corrected. My lab is studying many key questions about chromosome segregation, including how kinetochores assemble, how kinetochores make bioriented microtubule attachments, and how the spindle checkpoint detects and corrects defects in these processes.

One of our interests is a key regulator of chromosome segregation in all eukaryotes, the Ipl1/Aurora protein kinase. Because the Aurora kinases are oncogenes and amplified in many tumor types, studies on the budding yeast Ipl1 protein are critical to understanding both chromosome segregation and the maintenance of genomic stability. We previously found that Ipl1 leads to the detachment of mono-oriented kinetochores that are not under tension, giving the cell a chance to make proper bioriented attachments to kinetochores (Pinsky, B. A. et al., 2003). We recently showedthat Ipl1 is also required to activate the spindle checkpoint when there is a defect in kinetochore tension because it creates unattached kinetochores that signal to the checkpoint (Pinsky, B. A. et al., 2006). We have also discovered 3 additional Ipl1 functions that are likely related to a general role in regulating microtubules (Buvelot et al., 2003 and Kotwaliwale et al., 2007). Ipl1 is required for spindle breakdown, spindle positioning and spindle assembly. Although Ipl1 has many functions, all of them appear to be related to a common role in regulating microtubules. We are therefore performing assays to determine how Ipl1 alters microtubule dynamics and stability in vitro. In addition, we are analyzing the regulation of Ipl1 kinase activity and identifying its substrates to understand how it mediates microtubule destabilization in the presence of tension. Finally, we are studying the dynamic localization of Ipl1 that is likely related to its ability to carry out numerous functions.

We are also studying the functions and regulation of the centromeric histone H3 variant (CenH3) that likely forms a specialized centromeric nucleosome. Because CenH3 is associated with all active kinetochores, it may be the epigenetic mark that specifies the site of kinetochore assembly. We discovered that CenH3 is regulated by ubiquitin-mediated proteolysis and isolated dominant lethal Cse4 mutants that are resistant to proteolysis (Collins, K. A., et al., 2004). Stabilized CenH3 localizes to euchromatin, indicating that proteolysis helps to restrict CenH3 to kinetochores. We also showed that the a centromeric nucleosome is essential for de novo kinetochore assembly (Collins, K. A., 2005). We have recently developed a new assay to monitor Cse4localization at single nucleosome resolution and are using this technique to analyze the mechanism that restricts Cse4 to centromeric DNA (Furuyama and Biggins, 2007). In addition, we have initiated experiments to identify the Cse4 chaperone that targets it to the centromere.

In addition to continued studies on the above topics, we have recently developed a minichromosome purification to dissect the requirements for kinetochore assembly and function. Using this assay, we have identified new kinetochore proteins as well as a number of kinetochore post-translational modifications. In addition, we have initiated a screen to identify spindle repair systems that correct spindle defects. Finally, we are characterizing new chromosome segregation pathways. We have identified many non-essential genes that are synthetically lethal when deleted in an ipl1 mutant strain. Characterization of these genes will identify pathways that act in parallel with Ipl1 to regulate chromosome segregation. Taken together, these studies should continue to elucidate new details about the mechanisms of chromosome segregation and thus aid in understanding the generation of aneuploidy and disease progression.