Mechanosensitive signaling of the Spindle Assembly Checkpoint

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 DESCRIPTION (provided by applicant): The Spindle Assembly Checkpoint (SAC) is a signaling mechanism that ensures accurate chromosome segregation during cell division. It is known that the SAC is exquisitely sensitive to the state of attachment of each kinetochore to spindle microtubules. However, the molecular mechanism that enables this `mechanosensitive' signaling remains a central mystery in cell biology. We propose that the kinetochore encodes a mechanical switch consisting of two protein `terminals'. The physical separation of these protein terminals, akin to the opening of a switch, is necessary for disrupting the biochemical signaling cascade of the spindle SAC. This proposal will define the biochemical activities and structural properties of the constituent proteins that enable the kinetochore to behave as a mechanical switch. We will first define the molecular details in the simple budding yeast kinetochore (Aim 1), and then extend key experiments to the human kinetochore using HeLa cells (Aim 2). This work will establish the basis of mechanosensitive SAC signaling in eukaryotes. We will also engineer a novel, biochemical `potentiometer' that quantifies the signaling potential of the SAC (Aim 3). The signaling potential, defined as its ability of the SAC to induce and sustain cell cycle arrest, plays a key role in development, proliferation of aneuploid tumor cells, and age-related infertilit. The potentiometer will be a much-needed and versatile tool for investigating these critical aspects of cell biology.