Functional Intersection of the Spindle Assembly Checkpoint and the Kinetochore

纺锤体组装检查点（SAC）协调细胞周期进程与有丝分裂纺锤体组装。 未连接或不正确连接到纺锤体的动粒产生抑制细胞周期进程的信号，在中期-后期过渡。PI正在研究动粒蛋白和SAC途径之间的功能交叉。 研究表明，S.酿酒酵母SAC蛋白激酶Bub1除了需要产生纺锤体损伤信号外，还起着指定正常动粒结构和功能的作用。 动粒作用通过其与着丝粒特异性组蛋白H3变体Cse4（a.k.a. CENP-A或CenH3）。 与其他研究小组一起，该实验室发现了Cse4的另外两个不同的N-末端区域：一个与保守的有丝分裂调节激酶波罗（酵母中的Cdc 5）相互作用，另一个与Ctf 19动粒蛋白复合物相互作用。 中心假设是Cse4 N-末端结构域作为这些因素之间的关键功能相互作用的平台，其影响SAC响应和动粒功能。 主要研究者将对单个细胞类型S内关键的着丝粒相关因子（Cse 4/CENP-A、Bub 1、Cdc 5/波罗、Ctf 19复合物等）之间的各种相互作用进行系统分析。啤酒。 本研究的主要目的是：1）确定SAC及其相关蛋白在动粒上的组装途径。我们将利用S。酿酒酵母实验系统，系统和定量分析如何一个复杂的相互作用的关键因素与动粒。2)以确定Ctf19复合物的动粒和SAC作用。 该复合物与SAC功能、从头动粒建立有关，并且发现对于募集粘着蛋白和关键SAC调节剂极光B激酶两者都很重要。我们已经发现了这11个亚基复合物的三个结构分支的证据。 该复合物或其分支所起的作用将通过观察功能丧失的后果来评估。3）确定Cdc 5（波罗激酶）的动粒和SAC作用。 波罗与脊椎动物细胞中的SAC反应有关，然而，没有研究提供SAC反应实际上需要波罗激酶的证据。 这里的重点将是确定角色，SAC或其他，Cdc5在着丝粒中执行。Cdc 5和CEN定位的粘附素之间的新观察到的联系也将被探索。4）检查Sgo 1/Shugoshin和Ipl 1/Aurora B的动粒作用。 这些因素激活SAC反应，特别是在姐妹着丝粒之间没有正常双极张力的情况下。 计划中的实验将揭示他们的着丝粒招聘机制，以及它们如何影响其他着丝粒活动。更广泛的影响：本科生将执行许多所描述的实验。 PI是本科实验室研究的强烈支持者。事实上，四名大学生收集了这里提供的大部分初步数据。 PI与约翰霍普金斯大学本科生有着广泛的联系（超过80个课时/年），在两个生物学主要核心课程中授课：遗传学（160名学生/年）和细胞生物学（350名学生/年）。许多学生参加约翰霍普金斯，因为它的研究声誉，但良好的实验室经验以外的课堂可能很难为他们获得。 PI计划提供这个项目的学习机会，作为学分的本科研究课程。 学生将参加每周会议，讨论理论，策略和协议。 学生也将在这些会议上介绍他们的进展。本文描述的许多实验技术，虽然具有挑战性，但将迭代执行，因此将是本科生的优秀实验室培训经验。

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.