Developing an In Vivo Assay for Quantitative Analysis of Arp2/3 Complex Inhibitors
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Topping, Maisie
Narvaez Ortiz, Heidy
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Abstract
Branched networks in the actin cytoskeleton are critical for a variety of cellular processes such as motility and endocytosis. New branched actin filaments are nucleated by Arp2/3 complex, and the deregulation of this protein is related to a variety of diseases including cancer. Several classes of small organic molecule inhibitors of Arp2/3 complex have been discovered, most of which function by blocking an activating conformational change of the complex. These molecules serve as useful research tools because they allow researchers to turn off activity in many different processes, and they also have potential as drugs due to Arp2/3 complex’s increased activity in disease mechanisms. These inhibitors have been characterized in vitro and have been used in many experiments, but they have never been quantitatively analyzed in vivo. My project will develop an in vivo assay for quantitatively measuring the effects of Arp2/3 complex inhibitors on cytoskeleton dynamics. The assay will use Drosophila S2 cells expressing a low level of GFP-tagged actin and total internal reflection fluorescence (TIRF) microscopy to extract velocity data from the cell’s actin cytoskeleton before and after treatment with inhibitors. These experiments will lead to a better understanding of how Arp2/3 complex inhibitors affect living things, because this assay is a better approximation of biological systems than the currently used in vitro methods. The different assays can be used in concert to provide a fuller characterization of the inhibitors than we currently have.
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Single page poster
Keywords
biochemistry, cytoskeleton, inhibitors, microscopy