This proposal aims to study the mechanism by which kinetochore proteins capture microtubules to ensure equal chromosome segregation and timely progression through mitosis. Through analyzing the spindle-associated lamin-B containing network, which we had referred to as the spindle matrix previously, we have made a number of exciting observations, which suggest that component(s) of the lamin-B network functions together with microtubules as chaperones for kinetochore proteins such as Bub3. These chaperones are required for kinetochore proteins to regulate proper microtubule and kinetochore interactions and timely mitosis progression. We propose to dissect the molecular mechanism by which these chaperones function in mitosis. Specifically, in Aim 1 we will dissect whether and how the interaction between BuGZ and microtubules and/or EB1 is required for Bub3 kinetochore loading and chromosome alignment.
In Aim 2 we will study whether the destabilization of BuGZ and Bub3 by RanGTP contributes to silencing of the spindle assembly checkpoint (SAC).
In Aim 3, we propose to study the molecular mechanisms that mediate end-on kinetochore-MT attachment and SAC silencing.

Public Health Relevance

Extensive effort has been devoted to deciphering how microtubules capture kinetochores to ensure equal segregation of chromosomes and timely mitotic exit. Despite the discovery of many molecules involved in kinetochore assembly and kinetochore-microtubule interactions, how the dynamic kinetochore proteins change from binding along the sides of MTs (side-on interactions) initially to binding to the plus ends of MTs (end-on interactions) has remained unclear. Since the end-on binding is essential for equal chromosome segregation, understanding the molecular mechanism is critical. The current models explaining how kinetochores establish interaction with microtubules have focused on the kinetochore proteins required for binding to microtubules. Although microtubule dynamics are important in this process, microtubules are considered a passive recipient of the kinetochore microtubule capture machinery. Considering the dynamic behavior of kinetochore proteins in mitosis, we reasoned that proteins associated with the spindle could function to chaperone kinetochore protein loading to kinetochores and their capture of microtubules. By analyzing the proteome of the spindle-associated lamin-B-containing network isolated from Xenopus egg extracts, we have uncovered new players involved in chaperoning kinetochore-microtubule interactions. Our observations show that microtubules actively participate in the assembly of the microtubule capture machinery at the kinetochore. We propose to further dissect the mechanism. Although kinetochore-microtubule interaction is an extensively studied topic in mitosis, by looking outside of the kinetochores we have gained the opportunity to elucidate the key mechanism that has not been considered previously. We believe that pursuing the aims proposed in this grant will provide new avenues to explore therapeutic targets for halting uncontrolled cell proliferation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM110151-04
Application #
9412487
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Deatherage, James F
Project Start
2015-04-01
Project End
2019-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Carnegie Institution of Washington, D.C.
Department
Type
DUNS #
072641707
City
Washington
State
DC
Country
United States
Zip Code
20005
Gigante, Crystal M; Dibattista, Michele; Dong, Frederick N et al. (2017) Lamin B1 is required for mature neuron-specific gene expression during olfactory sensory neuron differentiation. Nat Commun 8:15098
Wan, Yihan; Zheng, Xiaobin; Chen, Haiyang et al. (2015) Splicing function of mitotic regulators links R-loop-mediated DNA damage to tumor cell killing. J Cell Biol 209:235-46
Jiang, Hao; He, Xiaonan; Feng, Di et al. (2015) RanGTP aids anaphase entry through Ubr5-mediated protein turnover. J Cell Biol 211:7-18