The role of ZW10 and its binding partners in chromosome segregation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.Kinetochores are proteinacious structures that assemble on centromeric DNA and fulfill several important functions during chromosome segregation. They attach chromosomes to microtubules of the mitotic spindle in a bipolar fashion and monitor the state of the kinetochore-microtubule attachment. In case errors are present, a monitoring mechanism, the spindle assembly checkpoint, inhibits metaphase to anaphase transition until proper bipolar attachments are formed. The mechanics of the attachment process is poorly understood and functions of many players involved are largely unknown. The relationship between the formation of kinetochore-microtubule attachment and its monitoring by the checkpoint remains unclear. I combined an RNAi technology with the high resolution microscopy to analyze the function of six microtubule-binding proteins and a checkpoint protein complex ROD/ZW 10/Zwilch (RZZ) in chromosome segregation and spindle checkpoint signaling. I discovered a non-redundant role for CLIP- 170, dynein/dynactin, LIS 1 and TOG I in chromosome congression to the metaphase plate. Selective depletion of dynein/dynactin and CLIP- 170 from mitotic kinetochores by RNAi of ZW 10, uncovered a novel role for these proteins in the initial kinetochore-microtubule encounter. Surprisingly, my results also demonstrate that ZW10 functions in the spindle checkpoint signaling independently of previously proposed downstream player, Mad2. In addition, I identified a ZW10's interaction with BubRI that may suggest a possible role for ZW 10 in the checkpoint signaling pathway. Thus RZZ complex acts at the interphase of attachment and signaling at kinetochores suggesting a close link between structural establishment of kinetochore-microtubule binding and its monitoring by the spindle checkpoint, in contrary to classical thinking.by Irina Michailovna Shapiro.Ph.D