The spindle assembly checkpoint (SAC) coordinates cell cycle progression with mitotic spindle assembly. Kinetochores that are unattached or improperly attached to the spindle generate a signal that inhibits cell cycle progression at the metaphase-anaphase transition. The PI is investigating the functional intersection between kinetochore proteins and the SAC pathway. The research has demonstrated that the S. cerevisiae SAC protein kinase Bub1 acts to specify normal kinetochore structure and function, in addition to its requirement for generating the spindle-damage signal. The kinetochore role is accomplished via its association with the N-terminus of the centromere-specific histone H3 variant Cse4 (a.k.a. CENP-A or CenH3). Together with other research groups, this laboratory has found two other distinct N-terminal regions of Cse4: one interacts with the conserved mitotic regulatory kinase Polo (Cdc5 in yeast) and the other with the Ctf19 kinetochore protein complex. The central hypothesis is that the Cse4 N-terminal domain serves as a platform for key functional interactions between these factors that both affect the SAC response and kinetochore function. The PI will perform a systematic dissection of the various interactions between key kinetochore-associated factors (Cse4/CENP-A, Bub1, Cdc5/Polo, Ctf19 complex, and others) within a single cell type, S. cerevisiae. The overall goal is a precise description of how they act in concert to accomplish the SAC response and kinetochore functions.
The Specific Aims of this project are: 1) to determine the assembly pathway(s) of key SAC and related protein onto the kinetochore. We will take advantage of the S. cerevisiae experimental system to systematically and quantitatively analyze how a complex interacting set of key factors associates with the kinetochore. 2) to determine the kinetochore and SAC role of the Ctf19 complex. This complex has been implicated in SAC function, de novo kinetochore establishment and found to be important for recruiting both cohesin and the key SAC regulator Aurora B kinase. We have found evidence for three structural branches to this 11 subunit complex. The roles this complex, or branches of it, perform will be assessed by looking at the consequences of loss-of-function. 3) to determine the kinetochore and SAC role of Cdc5, the Polo kinase. Polo has been implicated in the SAC response in vertebrate cells, however, no study has provided evidence that the SAC response actually requires Polo kinase. The focus here will be determining the role(s), SAC or otherwise, Cdc5 performs at kinetochores. A newly observed link between Cdc5 and CEN-localized cohesin will also be explored. 4) to examine the kinetochore roles of Sgo1/Shugoshin and Ipl1/Aurora B. These factors activate the SAC response specifically in the absence of normal bipolar tension across sister kinetochores. The planned experiments will reveal the mechanism of their kinetochore recruitment and how they influence other kinetochore activities.
Broader impact: Undergraduate students will perform many of the described experiments. The PI is a strong proponent of undergraduate lab research. Indeed, four undergraduates gathered much of the preliminary data presented here. The PI enjoys extensive contact with Johns Hopkins undergraduates (over 80 lecture hours/year), teaching in two Biology major core classes: Genetics (160 student/year) and Cell Biology (350 students/year). Many students attend Johns Hopkins because of its reputation for research, yet good lab experiences outside of the classroom can be difficult for them to obtain. The PI plans to offer study opportunity on this project as an undergraduate research course for academic credit. Students will attend weekly meetings to discuss theory, strategies and protocols. Students will also present their progress at these meetings. Many of the experimental techniques described herein, while challenging, will be performed iteratively and are thus will be excellent lab training experiences for undergraduate students.
Proper mitotic cell division requires the action of the kinetochore, a specialized chromatin site that accomplishes mechanical and regulatory processes that are essential for chromosome segregation. The best characterized kinetochore is that of the yeast S. cerevisiae, a structure consisting of ~80 proteins that link each centromeric DNA to a single spindle microtubule. These proteins form smaller sub-complexes that associate with centromeric DNA in a hierarchical manner. The bulk of our studies in the award period were devoted to the understanding of the assembly and function of two key kinetochore proteins, the CTF19 complex and Cdc5 (a Polo-like kinase). The central kinetochore CTF19 complex is important for proper kinetochore function and has been shown to participate in various kinetochore activities such as chromosome biorientation, recruitment of centromere-localized cohesion and recruitment of other key proteins that act to regulate mitotic spindle function. CTF19 consist of 2 protein subunits essential for yeast cell viability plus 9 additional nonessential subunits. Studies by others showed that the CTF19 "core" consists of the 2 essential subunits plus 2 nonessential. Our studies, supported by this award, demonstrated that the remaining 7 nonessential subunits independently assemble into three distinct branches (see Figure 1). The three branches seem to have different functions as well, although more research is required to define their exact roles. Polo-like kinases (Plk’s) drive numerous key regulatory steps throughout the M-phase of the eukaryotic cell cycle. Some of these steps appear to be accomplished by the Plk molecules specifically localized at kinetochores. We developed a novel assay that allowed us to examine the association of Cdc5, the Plk in S. cerevisiae, with kinetochores and other chromosomal sites. We found that Cdc5 associates at sites that have recruited cohesin, the "glue" that holds sister chromatids together until anaphase. Kinetochore association of Cdc5 was dependent upon cohesin, but not the reverse. In the absence of Cdc5, cohesin persisted at kinetochores into anaphase, consistent with the known role of Plk in cohesin removal. However, under these conditions chromosomes were able to separate, despite high cohesin at kinetochores, suggesting that we have discovered a new form of cohesin that can associate, but has lost its chromatid "glue" function. Our findings will significantly add to the body of knowledge concerning kinetochore assembly and function.
