Tag: Brad Nolen

Presenter: Maisie Topping – Biochemistry

Faculty Mentor(s): Brad Nolen, Heidy Narvaez Ortiz

Session: (In-Person) Poster Presentation

Branched networks in the actin cytoskeleton are critical for a variety of cellular processes including endocytosis. New branched actin filaments are nucleated by Arp2/3 complex, and the deregulation of this protein is related to diseases such as cancer. Arp2/3 complex is intrinsically inactive. During activation, the complex undergoes a conformational change that brings two of its subunits, the actin-related proteins Arp2 and Arp3, into a position that mimics two consecutive actin subunits within a filament, thereby creating a template for the new filament. When actin polymerizes into filaments, a portion of the protein called the D-loop helps to stabilize the filamentous structure, and the Arp2 and Arp3 subunits both contain a similar D-loop. A previously solved structure of Arp2/3 complex at a branch junction indicates that a contact between the D-loop of Arp2 and ArpC3 may be important for stabilizing the activated complex at the junction site. This project aimed to assess the importance of that contact in activation of Arp2/3 complex. We generated a strain of budding yeast with three mutations in the Arp2 D-loop, purified Arp2/3 complex from cells, and used pyrene actin polymerization assays to test the ability of the mutated complex to nucleate actin filaments compared to the wild type. The Arp2 triple mutant showed greatly decreased activity, indicating that the contacts between Arp2 and ArpC3 are important for the activation and function of Arp2/3 complex.

Presenter: Maisie Topping – Biochemistry

Faculty Mentor(s): Brad Nolen, Heidy Narvaez Ortiz

Session: (In-Person) Poster Presentation

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 molecule inhibitors of Arp2/3 complex have been discovered, most of which function by blocking an activating conformational change of the complex. These molecules are useful tools because they allow researchers to turn off activity in different processes, and they have potential as drugs due to Arp2/3 complex’s increased activity in some diseases. These inhibitors have been characterized in vitro and have been used in 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 these inhibitors.