Defining the role of Arp2/3 complex diversity at multiple scales of biology

The actin cytoskeleton of the cell is critical for the complex, integrated processes associated with development, operation and sustainability of the human body. The Arp2/3 complex consisting of seven protein subunits is essential to stimulate dynamic branched actin networks needed for multiple cellular processes. The Arp2/3 complex has always been considered as a single entity, but in humans and other mammals, three of the Arp2/3 complex subunits are encoded by two isoforms, thus allowing the formation of eight distinct Arp2/3 complexes. The Way lab has shown that Arp2/3 subunit composition dramatically affects the formation and stability of branched actin networks. The Way and Gomes labs have shown how specific Arp2/3 isoforms are essential for muscle development.Our synergistic, high-risk, high-gain goal is to define the role of Arp2/3 complex diversity at three hierarchies of biology:1. Molecular basis of Arp2/3 diversificationWith purified isoform-specific complexes we will perform cryo-electron microscopy and single molecule fluorescence microscopy to reveal the structural variations and influence of Arp2/3 diversity on actin networks in vitro.2. Cellular function of different Arp2/3 complexesWith cells expressing specific Arp2/3 isoforms, we will use quantitative live cell imaging and cryoelectron tomography to reveal the dependence of cellular actin networks on Arp2/3 diversity and its functional consequences.3. Developmental and physiological role of individual Arp2/3 complexes. With genetically modified cultured myofibers and transgenic mice, we will use an array of imaging approaches to reveal the contribution of different Arp2/3 family members to muscle development, structure and physiology.Our interdisciplinary plan builds on the strengths of our three labs, takes advantage of unique reagents and powerful model systems, and will allow us to determine how Arp2/3 diversity contributes to biological complexity at multiple scales.