The role of the kinetochore in chromosome signaling and segregation

<p>Error-free chromosome segregation is crucial for the maintenance of genomic stability and</p> <p>prevention of tumorigenesis. The spindle assembly checkpoint (SAC) prevents exit from</p> <p>mitosis as long as not all chromosome pairs are properly attached to the mitotic spindle.</p> <p>Microtubules attach to chromosomes through binding at the kinetochore, a multi-protein</p> <p>structure that is organized over centromeric chromatin. The kinetochore plays an</p> <p>important role in the generation of the SAC signal that prevent mitotic exit while at the</p> <p>same time it is involved in chromosome movements during congression and segregation.</p> <p>The work reported in this thesis aims to further our understanding on how the combination</p> <p>of such functions ensures faithful chromosome segregation. The first part focuses on the</p> <p>phenotypic analysis of Drosophila melanogaster S2 cells depleted of the SAC protein</p> <p>BubR1 and the microtubule motor protein CENP-meta/CENP-E. The results show that</p> <p>while BubR1-depleted cells exit mitosis prematurely due to loss of SAC activity, CENPmeta-</p> <p>depleted cells are delayed in mitosis. Furthermore, a detailed analysis of</p> <p>microtubule-kinetochore interactions after depletion of BubR1 or CENP-meta clearly</p> <p>shows that without BubR1 cells contain a high frequency of monoriented and fully</p> <p>unattached chromosomes while most CENP-meta-depleted cells have chromosomes</p> <p>attached to spindle microtubules. Moreover, when both proteins are simultaneously</p> <p>depleted absence of CENP-meta is able to partly rescue microtubule-kinetochore</p> <p>attachments lost due to BubR1 depletion. The data shows that BubR1 is required to</p> <p>promote stable microtubule-kinetochore attachment. However, CENP-E appears to</p> <p>promote kinetochore-microtubule instability, suggesting a role of CENP-E in the</p> <p>mechanism that corrects inappropriate kinetochore attachment. Accordingly, the second</p> <p>part of the thesis concentrates in determining whether CENP-E and Aurora B kinase</p> <p>activities cooperate to promote efficient chromosome biorientation. The results, obtained</p> <p>in HeLa cells, do not support a regulation of Aurora B kinase activity by CENP-E.</p> <p>However, strongly suggest that Aurora B kinase destabilizes spindle pole proximal</p> <p>kinetochore-microtubule interactions keeping the SAC active to allow CENP-E-mediated</p> <p>congression of monoriented chromosomes. Taken together, the work presented in this</p> <p>thesis, through the study of two bona fide kinetochore proteins, provides insight in how</p> <p>chromosomes congress and how can they delay mitotic exit when errors occur thus</p> <p>avoiding aneuploidy.