Microtubule dynamics and error correction

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Project Summary This project requests funding to upgrade equipment to clone and analyze CRISPR/Cas9 transgenic recombinant cells. These cells are required to address the Specific Aims in NIH grant R01GM145567. The broad goal of this project is to understand the mechanistic contribution of force generation by kinesin-related motors and dynamic microtubules to chromosome segregation. To accomplish these goals we engineer CRISPR cells with fluorescently tagged kinesin motors that can be rapidly relocated to diverse regions of the cell. We have found that functional analysis of proteins in these cells is much more physiologically relevant than the more commonly used transfection and expression methods. This is in part because the proteins are at the appropriate level in the cell and in part because there is no contaminating activity from endogenous protein because the modifications for visualization and experimental manipulation are performed on the endogenous gene. One disadvantage is that the physiologically relevant levels of protein is challenging to image in cells. Our isolation and analysis of these cells is performed microscopically and critically dependent on our imaging equipment. To isolate and functionally evaluate these cells we use a custom-modified high resolution, light sensitive GEHealthcare microscopy system. Unfortunately, the company that previously covered its service contract (GEHealthcare/Cytiva) no longer exists as a microscope company. Thus, there are no parts available to cover instrument failures. As of 9/12/22 the instrument controller has failed leaving us without the key instrument underscoring our productivity for live cell imaging. We have devised a means to cannibalize the working parts of our system and combine it with a minimal version of the Nikon CREST X-Light V3 spinning disk confocal. This system will have two advantages: 1) it will reinstate our imaging capabilities and, accordingly, our scientific productivity; and 2) it will allow us to image both flat cultured cells and embryonic zebrafish. This will provide us with both additional capability to investigate the contribution of poorly expressed kinesins (such as Kif9 and Kif25) to organismal development, per our approved change of scope addition of Danio rerio, and restore our ability to isolate and evaluate CRISPR-engineered cell lines.