טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTeichner Adar
SubjectCharacterization of Different Inhibitors of APC/C in
the Mitotic Checkpoint
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The mitotic (or spindle assembly) checkpoint system is a surveillance mechanism that prevents the initiation of anaphase until all sister chromatids are correctly attached to the mitotic spindle through their kinetochores. It thus acts as a safeguard mechanism to ensure the accuracy of chromosome segregation in mitosis. The target of the mitotic checkpoint system is the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that acts on some cell cycle regulatory proteins such as mitotic cyclins and securin, an inhibitor of anaphase initiation. When the checkpoint system is switched on, it inhibits the APC/C, securin cannot be degraded and anaphase cannot be initiated. Although much genetic information is available on the identity of components of the mitotic checkpoint system, the biochemical mechanisms by which it regulates the activity of APC/C remained obscure. An exception was the identification of a mitotic checkpoint complex (MCC), an APC/C inhibitor that is composed of the APC/C activator Cdc20, associated with the checkpoint proteins Mad2, BubR1 and Bub3. The MCC assembles during checkpoint and inhibits the activity of APC/C. However, it is not known what are the molecular mechanisms of the assembly of MCC when the checkpoint system is turned on, and of the disassembly of MCC when the checkpoint is turned off. It was also not known whether there are other checkpoint inhibitors of APC/C (in addition to MCC), since in many other cases several parallel mechanisms exist to control an important cellular process.

   Previous work from our laboratory has used extracts from nocodazole-arrested cells to gain insight into some biochemical mechanisms of the mitotic checkpoint system. Nocodazole prevents the assembly of the microtubules of the mitotic spindle and thus produces a strong checkpoint signal. Soluble extracts from nocodazole-arrested cells do not contain chromosomes, but they can be used to study downstream events of the mitotic checkpoint system, such as the nature of the mitotic checkpoint inhibitors and the mechanisms of the disassembly or inactivation of these inhibitors in exit from checkpoint-arrested state. The aim of this research was to gain knowledge of two related problems: (1) What are the identity and characteristics of different mitotic checkpoint inhibitors of APC/C? (2) What are the molecular mechanisms of the disassembly of one of these inhibitors, MCC, in release from mitotic checkpoint?