Reconstitution of the load-bearing attachments between the human kinetochore and microtubule ends

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Project Summary When a cell divides it must accurately segregate its duplicated chromosomes between the two daughter cells. Errors in the function or regulation of chromosome segregation can produce less viable cells with extraneous chromosomes. Interestingly, extra and unstable chromosomes are often found in cancer cells but it is unknown if genome damage created by these segregation errors can promote cancer formation or growth. To understand the impact of these abnormal genomes in cancer and other disease progression it is essential to determine how chromosome segregation occurs under normal conditions and the steps at which this process can malfunction to produce damaged genomes. The kinetochore is a ~100 component molecular machine that connects microtubule ends to chromosomes and harnesses the power of depolymerizing microtubules to segregate chromosomes. Importantly, kinetochores must maintain their connection to chromosomes and microtubule ends while under high tension. Weakening kinetochore microtubule connections can halt cell division or promote incorrect chromosome segregation. While the kinetochore components that bind microtubules are mostly determined, it is still unknown how these components coordinate to bear the forces necessary to regulate and perform chromosome segregation. For this proposal, I will use purified components to reconstruct the minimal human kinetochore microtubule interface at its native attachment strength. I will accomplish this by testing the load-bearing strength of different combinations of purified kinetochore components at microtubule ends. Additionally, I will test if multiple copies of the binding components strengthen microtubule attachment by assembling into a specific geometry or increasing the number of binding interactions. Attachment strength will be measured using an optical trap to precisely manipulate and measure the forces experienced by the kinetochore components bound to microtubule ends. My reconstitution of the kinetochore microtubule attachment interface will establish a minimal system by which to test the role of load-bearing strength in the function and regulation of cell division. Importantly, this reconstituted system provides a means by which to examine native kinetochore microtubule attachments in a highly controllable environment outside of cells. Increasing the complexity of this system by introducing regulatory signaling molecules will test how microtubule kinetochore attachments control the progression of cell division. Identification of error-prone interactions within the regulatory pathways of chromosome segregation will promote the development of novel experiments into the role of cell division errors in cancer and other diseases. Understanding how significant aberrant genomes form is important to improving our identification, prevention and treatment of damaged genome diseases, such as cancer.