Multiple domains in the NDC80 complex are required for generating and regulating kinetochore-microtubule attachments in mitosis
Date
2012
Authors
Sundin, Lynsie, author
DeLuca, Jennifer G., advisor
Bamburg, James, committee member
Curthoys, Norman, committee member
Bailey, Susan, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The goal of mitosis is to accurately segregate chromosomes into two new daughter cells. It is critical that this process occurs appropriately because the consequences of chromosome nondisjunction or missegregation are severe, most notably birth defects and cancer. Kinetochores are built at the centromeric region of mitotic chromosomes and serve several functions during mitosis. First, the kinetochore is the physical scaffold at which microtubule binding sites are built. Second, kinetochores regulate the strength of the attachments to microtubules to ensure proper chromosome movements. Finally, the kinetochore is the origin of a soluble 'wait anaphase' signal that prevents premature entry into anaphase. Together these functions culminate with chromosome alignment at the spindle equator of a cell, ultimately resulting in accurate chromosome segregation in anaphase. While the kinetochore can be considered the director of kinetochore-microtubule attachment, microtubules drive the process of cell division by providing the force behind chromosome movements. The mechanism of kinetochore-microtubule attachment remains elusive as kinetochores must generate and maintain connections to microtubules that are constantly polymerizing and depolymerzing. Extensive studies into this process have revealed that the KMN (KNL1 complex, MIS12 complex, and NDC80 complex) network, a supercomplex of proteins at the outer kinetochore, comprises the core microtubule binding site in cells. As part of this network the NDC80 complex has been an attractive candidate as an essential part of the microtubule binding machinery. Here we have used a combination of in vivo, in vitro, and in silico methods to characterize three discrete domains of the NDC80 complex that each contribute to the process of kinetochore-microtubule attachment in distinct ways. Our data have elucidated some of the molecular details of how kinetochore-microtubule attachments are both generated and regulated. We show that the Hec1 CH domain is absolutely required for kinetochore-microtubule attachment. Our data suggest that the Hec1 CH domain makes direct contacts with microtubules, while the CH domain of Nuf2 does not, indicating functionally distinct roles for these protein domains in mitosis. We characterize the Hec1 loop domain, demonstrating that it is required for stable kinetochore-microtubule attachments and mitotic progression. Our data suggest that the Hec1 loop domain is required to recruit accessory proteins to the kinetochore during mitosis. Furthermore, we show that kinetochore-microtubule attachment strength is highly sensitive to small changes in Hec1 tail phosphorylation. Finally we also demonstrate that incremental phosphorylation of the Hec1 tail domain is a primary mechanism of regulating kinetochore-microtubule attachment strength. Together our data highlight the diverse functions of a single kinetochore component and implicate the NDC80 complex as the principle site for direct binding to microtubules and as a site of regulation for these attachments.
Description
Zip file contains movie legend and supplementary videos.
Rights Access
Subject
cell cycle
kinetochore
NDC80
microtubule
mitosis