Kinetochore Specification and Function

点击翻译按钮获取中文摘要

PROJECT SUMMARY For the development of an organism from a single cell, the genome must be duplicated and precisely distributed during every cell division. Given the astronomical number of cell division events involved in building and maintaining organisms (by one estimate, the adult human body is made up of 40 trillion cells, with at least a couple of trillion dividing every day), it becomes critical that the distribution of the replicated genome to daughter cells, a process known as chromosome segregation, occurs with extremely high accuracy. Errors in chromosome segregation underlie birth defects/infertility and are sufficient to trigger the genomic havoc that is a hallmark of cancer. Thus, understanding the mechanisms by which cells ensure accurate chromosome segregation during cell division has both fundamental and therapeutic significance. Our work is focused on kinetochores, protein machines that assemble on mitotic chromosomes to orchestrate their segregation. Kinetochores coordinate multiple microtubule-interfacing activities that collectively orient and segregate chromosomes, while also integrating mechanical events with signaling mechanisms that control the decision to either remain in or exit from mitosis. Our early work identified a conserved set of proteins, referred to as the KMN network (for Knl1 complex, Mis12 complex, Ndc80 complex) that lies at the heart of these coordinated kinetochore functions. In Aim 1, we focus on the ability of kinetochores to both accelerate and delay exit from mitosis, which we propose optimizes mitotic duration to allow sufficient time for all chromosomes to connect to the spindle, while also minimizing time spent in a vulnerable phase when major cellular functions such as transcription, translation and secretion are downregulated. The work we propose tackles major open questions related to this duality of mitotic timing control by kinetochores. The primary mechanical activity of the kinetochore is to couple to dynamic spindle microtubules. Several distinct microtubule-interfacing activities concentrate at kinetochores as well as on mitotic chromatin, which makes analysis of chromosome-microtubule interactions challenging in a cellular context. In Aim 2, we describe a "blank slate–add back" approach, in which we eliminate all microtubule-interfacing activities on mitotic chromosomes and then add them back in isolation. This approach complements traditional loss-of-function analysis and will be used to study the microtubule-interfacing activities at kinetochores that orient and center chromosomes on the spindle and that shut of the signal that prevents mitotic exit until microtubule attachments are made. Finally, to achieve accurate segregation, kinetochore-microtubule interactions must be tightly regulated. In Aim 3, we focus on the poorly understood regulatory mechanism by which a spindle pole-generated gradient of the mitotic kinase Aurora A controls kinetochore-microtubule attachments. As Aurora A is amplified in specific cancer contexts, this effort has the potential to reveal cancer-specific dysregulation of chromosome segregation and suggest potential new therapeutic avenues.