Coupling kinetochore microtubule dynamics to chromosome motion Abstract: During cell division chromosomes must segregate equally to ensure the health and viability of the daughter cells. It is now well established that accurate chromosome segregation crucially depends on the force- transducing interactions between thread-like polymers (microtubules), and kinetochores, specialized chromosomal structures: when microtubules shorten, the chromosomes are transported to the opposite poles of a dividing cell. Loss of the proper connections between the kinetochores and shortening microtubules leads to a chromosome loss, and is one of the most significant causes of aneuploidy. However, the molecular mechanisms that ensure the stability of these dynamic connections are not known. Our long-term goal is to understand the fundamental biological functions: how the kinetochores of mitotic chromosomes are coupled to the dynamic microtubules ends and how these attachments remain stable under the load. In vitro, depolymerizing microtubules can move objects that are appropriately coupled to their shortening ends. Similar mechanisms are likely to play central role in the pole-directed chromosome movement. To study these processes in the quantitative and mechanistic way we have developed biophysical and single-molecule methods to dissect the interactions between isolated kinetochore proteins and dynamic microtubules in vitro under conditions that mimic aspects of normal kinetochore-microtubule attachments in cells. By using segmented polymers with photoliable plus-end caps we can trigger depolymerization in a highly controlled manner, which enables detailed analysis of disassembly-dependent forces. With these methods, here we seek to understand the molecular mechanisms of the microtubule-dependent coupling carried out by the essential human Ska1- complex, a presumptive functional homolog of the budding yeast Dam1.
Our Specific Aims are focused on determining the role of Ska1 oligomerization in assembling the microtubule tip-tracking structures and characterizing their ability to move processively with the shortening ends (Aim 1). We will critically examine how Ska1 captures the energy of microtubule depolymerization, and compare its efficiency with that of the Dam1 ring (Aim 2). To determine how purified Ska1 maintains stable attachment to the shortening polymer, we will use purified Ska1 to couple microtubules to glass microspheres, and examine their motions under a load applied with laser tweezers (Aim 3). This approach is innovative because it focuses sophisticated biophysical methodologies on specific coupling kinetochore complexes, which are essential for accurate inheritance of genetic information. This research is important because it will promote identification of the biomechanical features and specific protein modules that are responsible for a kinetochore's ability to slide along microtubule wall, to withstand counter-forces and to respond to maladaptive conditions in a noisy and stochastic environment of a dividing mammalian cell. Ultimately, this work will facilitate analysis of human diseases, such as cancer, in which accurate chromosome segregation fails.
The proposed research is relevant to public health because rigorous study of the dynamic linkages between spindle microtubules and chromosomal kinetochores will facilitate identification of the specific features of these force-transducing lins, which may be exploited for a selective disruption of kinetochore-microtubule interactions, thereby facilitating development of novel anti-cancer drugs. Thus, the proposed research is relevant to NIH's mission to foster fundamental creative discoveries that would ultimately advance our capacity to protect and improve human health.
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