CAREER: The Regulation of Cytokinesis by Calcium

University Of Toledo, Toledo OH

Investigators

Abstract

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117- 2). Millions of rounds of cell division are required for an embryo to become a fully developed human. How a cell can divide so reliably with precise timing and spatial cues is a fundamental biological question. One enduring mystery about cell division remains the involvement of calcium. We have long known that two quick currents consisting of calcium ions run through an embryonic cell during the last step of cell division called cytokinesis. These transient currents could potentially serve as the triggering signal, like a light switch, for the separation of two replicated daughter cells. However, their origin and function remain poorly understood due to technological challenges. This project will tackle this question by taking advantage of two fairly recent breakthroughs. The first is the conceptual advance of employing the simple fission yeast to study cytokinesis. This yeast is easy to manipulate genetically, but its cytokinesis is similar to that of the animal cells. The second is the technical advance of quantitative microscopy that can measure calcium current in live cells with high precision. The project will take advantage of these advances to uncover how calcium works during cytokinesis. Broader Impact activities include the intrinsic merit of the research as all eukaryotic cells likely utilize calcium signals during division. Additional activities involve the training of high school, undergraduate, and graduate students in research. The PI will extend the experience for high school students with remote controlled microscopes during the school year and he will also engage in activities to prepare and attract students for college. American Rescue Plan funding provides support for the investigator at a critical stage in his career. In this project, investigators will build on early advances to determine the mechanism of the calcium spikes. 1) The investigators will determine how the calcium spike arises in dividing fission yeast cells. We will test whether a special type of ion channels called mechanosensitive channels initiate the calcium spike. Then they will test whether two calcium pumps Pmr1 and Pmc1 clear out the excessive calcium so that the spikes are transient. 2) The investigators will determine how the calcium spike regulates the force-generator of cytokinesis the contractile ring. They will test whether the calcium spike promotes the activity of calcium-sensitive molecule Ppb1 which then proceeds to activate the motor protein type II myosins in the ring. They will measure how Ppb1 is recruited and activated in the ring and determine the activity and number of myosins in the mutant cells without Ppb1. This will be followed by the experiment to determine whether the myosin regulatory light chain is a substrate of Ppb1. 3) The investigators will determine how the calcium spike regulates the last step of cytokinesis the cell separation. They will determine whether the spike helps build the cell wall for yeast cells and maintain the integrity of the membrane through the activity of the calcium-sensitive molecule Cam1. They will employ yeast genetics, calcium imaging, atomic force microscopy and quantitative microscopy in the project. The investigators expect it will finally address the long-standing questions, where these cytokinetic calcium currents come from and what are their functions. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.