Amy Shaub Maddox Project Summary Our bodies are intricate ensembles of trillions of cells, and these building blocks are not generic like the bricks of a building, but rather are highly specialized. One characteristic of cells that distinguishes different types is their shape, from spherical, as in your circulating immune cells, to highly elongated, exemplified by the neurons that reach from our spines far into our legs. This project is aimed at understanding the mechanisms of cell shape change, which is crucial for many cell functions, including when a cell pinches into two after duplicating its contents. For 40 years, we have known that cell shape is maintained by some of the same cellular chemicals that cause our muscles to contract: actin, which forms long flexible polymers, and myosin, a biological motor that turns chemical energy into movement. Throughout these decades, substantial progress has been made in understanding how cells build actin polymers and turn on myosin motors, but less is known about how these essential events are attenuated. We reasoned that, just as the electronic governor on a heavy-duty truck limits its velocity, cells have ways of limiting actin-myosin work, to tune cell shape. In this project, we present several pieces of preliminary evidence of such cellular governors. We propose to determine the microscopic rearrangements that cause larger-scale cell shape changes, and define the chemicals that control these rearrangements. First, we demonstrate that the actin-myosin meshwork ring that pinches a cell in two does not go as fast as it can, but undergoes oscillations, speeding up and slowing down as it constricts. We routinely use powerful microscopes to observe living cells as they pinch in half, and measure the rearrangements that underlie these oscillations. Second, we will focus on the regulation of myosin, testing three distinct ideas about how its activity could be limited. Last, we present three new directions for the old field of studying cells pinching in half. To discover new candidate governors, we combined several technologies in ?fishing expedition? projects, which we have already completed. This unbiased but risky work laid well- substantiated groundwork for our functional studies of these new candidates. In sum, this project will take our field of cell pinching into novel territory, and will be relevant to the study of situations wherein cells purposefully arrest the pinching process, such as the generation of the specialized cells in your liver and heart muscle. Our findings will also contribute general principles of cell change shape, and thus help us understand normal development and diseases. Our discoveries may, in the long term, inform the development of therapies for pathologies involving cell shape regulation, such as cancers and blood diseases.