An In Vivo Assay for Quantitative Analysis of Arp2/3 Complex Inhibitors

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 assembly is linked to a variety of diseases including cancer. Several classes of small molecule inhibitors of Arp2/3 complex have been discovered and characterized in previous research. These molecules are useful tools because they allow researchers to turn off the activity of the complex in different processes, and they have potential as drugs due to the increased activity of Arp2/3 complex in some diseases. These inhibitors have been characterized in vitro and in some cell-based systems, but there has been limited in vivo quantitative analysis. My project developed an in vivo assay for quantitatively measuring the effects of Arp2/3 complex inhibitors on cytoskeleton dynamics. The assay uses 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 following treatment with inhibitors. Unlike previous experiments, this assay uses live cell imaging to directly measure actin dynamics in eukaryotic cells, and retains much of the complexity of biological systems including the presence of cell membranes and many different proteins. The previously used methods can be implemented in concert with this one to provide a more comprehensive characterization of these inhibitors.