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Title: Cellular and Molecular Analysis of Chromosomal Passenger Complex in Vertebrate Cells
Authors: Xu, Zhenjie
Supervisor(s): Earnshaw, William C.
Issue Date: 2009
Publisher: The University of Edinburgh
Abstract: Sucessful cell division is primarily controlled by temporal and spatial regulation of two critical events in the cell cycle: the faithful replication of chromosomes and the accurate segregation of the replicated chromosomes. Of all the cellular proteins involved in regulating these processes, the chromosomal passenger complex displays some of the most visually striking patterns of behaviours during mitosis. The chromosomal passenger proteins associate with chromosomes as cells prepare to segregate them during mitosis. Subsequently, when chromosomes are aligned on the spindle microtubules that do the mechanical work of segregation, the passenger proteins become concentrated in distinct foci between paired kinetochores—the protein-based structures on chromosomes where the microtubules attach. Then, as chromosomes start to segregate, the passenger proteins abruptly dissociate from the kinetochores and instead localize to the central spindle in the area midway between the separating chromosomes. This ultimately becomes the midbody during cytokinesis. The first protein found to display such dramatic changes in localization during nuclear division was named INCENP. Three further proteins—Aurora B, Survivin and Borealin were later found to show localization patterns similar to INCENP. Structural analyses revealed that these four proteins form a well conserved chromosomal passenger complex throughout mitosis, in which INCENP acts as a scaffold to recruit Survivin and Borealin to its N-terminus, while Aurora B associates with its C-terminus. The function of this complex parallels its dynamic localization. These include roles in spindle assembly, correcting erroneous kinetochore microtubule interactions that are inappropriate for chromosome alignment, signaling to the spindle checkpoint, and the completion of cytokinesis. Functional analysis of kinetochore proteins in vertebrates using DT40 cells has greatly improved our understanding of kinetochore assembly, kinetochore structure and functions in guiding cell division. Following the identification of chicken Aurora B, Borealin/Dasra B and Dasra A, we are now able to address the pleiotropic functions of the conserved chromosomal passenger complex in DT40 cells using gene disruption. Gene disruption of INCENP using a novel promoter hijack technique in DT40 cells has demonstrated that accurate cell division requires coordination of spindle dynamics, chromosome segregation and cell cleavage and that INCENP integrates these diverse processes. Recent concentrated efforts on elucidating the precise contribution of each subunit in localizing the complex have provided great insights of how CPC executes its mitotic function. Here, through introducing a series of INCENP structural mutants into this sophisticated INCENP knockout system, my in vivo data presented here indicated that interaction between INCENP-Aurora B is essential for cell division. Remarkably, dissociation of Aurora B from INCENP does not disturb the other passengers targeting to the centromere, but Aurora B activity through binding and activation by INCENP plays a direct role in guiding the transfer of the complex to the spindle midzone. Furthermore, a feed-back loop involving phosphorylation of INCENP at its TSS motif by Aurora B is essential for cell viability, even though its disruption by mutation to TAA does not prevent the passengers eventually transfer to spindle midzone and midbody. Thus, this final step to maximize the activity of Aurora B is a critical function of INCENP. The outstanding question I have addressed in my study is why the passenger proteins are interdependent. More importantly, my work reveals that the intrinsic modulation within chromosomal passenger complex directed by the interactions between INCENP and Aurora B regulates the transfer of the complex to the spindle midzone and midbody. Thus, the results here with previous study suggest that regulation of the centromere and spindle midzone targeting of the complex involves two distinctly intrinsic mechanisms directed by two targeting sub-complexes within CPC, in which INCENP, Borealin and Survivin form the core sub-complex for centromere targeting, and Aurora B regulated by INCENP forms the sub-complex serving to direct the CPC transfer to spindle midzone by modulation of Aurora B activity.
Keywords: Biology
Cell Biology
URI: http://hdl.handle.net/1842/3214
Appears in Collections:Biological Sciences thesis and dissertation collection

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