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Investigating the role of kinetochore dynein-dynactin in spindle assembly checkpoint function

Date

2020

Authors

Hodges, Amy Lauren, author
DeLuca, Jennifer, advisor
Markus, Steven, advisor
Bailey, Susan, committee member

Journal Title

Journal ISSN

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Abstract

When a cell divides it is essential that its chromosomes are equally divided into two new daughter cells, thus ensuring that each cell receives an identical copy of genetic information. The importance of this process is emphasized by the fact that a hallmark of cancer cells is erroneous chromosome segregation, leading to uncontrolled proliferation. A key cellular structure involved in maintaining genomic integrity is the kinetochore, a large proteinaceous structure that assembles upon centromeric chromatin during cell division. This complex structure is involved in linking mitotic chromosomes to spindle microtubules, as well as detecting and correcting erroneous kinetochore-microtubule attachments to ensure faithful chromosome segregation. Monitoring of kinetochore-microtubule attachments is carried out by the spindle assembly checkpoint (SAC), a surveillance system that generates a "wait anaphase" signal at unattached kinetochores, with the goal of delaying cell division until every kinetochore has attached to a spindle microtubule. The checkpoint signal is propagated by SAC effector proteins that accumulate at the outer surface of unattached kinetochores during mitosis. In metazoan cells, the minus end-directed motor protein cytoplasmic dynein-1 (dynein) is known to facilitate eviction of SAC effectors from the kinetochore upon stable microtubule attachment, effectively silencing the checkpoint and allowing for anaphase progression. It has been suggested that dynein-mediated eviction of checkpoint proteins is dependent on dynein's microtubule-based motor activity, with the prevailing model depicting SAC effectors transported as cargo toward the poles by the dynein motor along spindle microtubules. However, data supporting this model is lacking and the process is poorly understood. Here we have identified a subset of SAC effectors that require dynein for their removal from the kinetochore upon stable microtubule attachment. Additionally, we have generated a CRISPR cell line in which dynein is endogenously tagged, allowing us to characterize activity of kinetochore dynein in a manner not previously possible. Using this cell line in conjunction with small molecule-based inhibition of mitotic processes, we sought to investigate the role of spindle microtubules in dynein-mediated SAC silencing. Interestingly, our data show that dynein-mediated removal of key checkpoint proteins from kinetochores can occur in the complete absence of microtubules, suggesting a motility-independent role for the dynein motor in SAC silencing.

Description

2020 Fall.
Includes bibliographical references.

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Subject

kinteochore
spindle assembly checkpoint
mitosis
dynein

Citation

Associated Publications