Dissection of the coordinated regulation of actin and microtubule dynamics by mDia1 and APC in reconstituted systems by advanced TIRF microscopy and single-molecule analysis

• Applicant: Dr. Dennis Breitsprecher
• Subject Area: Biochemistry
• Term: from 2010 to 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 182105952

Cells have distinct shapes that can be rapidly remodeled by dynamic rearrangements of the cell’s ‘cytoskeleton’, which consists of a network of dynamic protein fibers. Two of its primary components are actin filaments and microtubules (MT), which have distinct properties and dynamics. For many years, the physiological functions of actin and MT were considered to be separate and controlled by distinct machinery and mechanisms. However, more recent evidence suggests instead that actin and MT functions are closely intertwined and must be tightly coordinated during cell morphogenesis and movement. The goal of this proposal is to gain new mechanistic insights in this area. Recent work in the Goode lab, where I will perform my research, has shown that the colon cancer-causing Adenomatous polyposis coli (APC) protein directly induces actin assembly in addition to its established roles in binding and stabilizing MT. This makes APC the first actin nucleator with direct links to cancer, and an ideal focal point for dissecting actin-MT cross-regulation. APC also exhibits a novel functional synergy in actin assembly with its in vivo binding partner, the formin mDia1, which also binds actin and MT. I will use advanced in vitro TIRF microscopy techniques to analyze the effects of APC and mDia1, alone and together, on the assembly of actin filaments and MT, and employ single molecule analysis of fluorescently labeled proteins to dissect the basis of APC/mDia1 synergy. These microscopy assays will be complemented by biochemical analyses of the mDia1-APC interaction and kinetic analyses of actin and MT polymerization in the presence of these proteins. I will also study regulation of these activities by EB1, another MT associated protein and in vivo binding partner of APC. This work will be beneficial to define a novel network of interactions and activities among APC, mDia1, and EB1 that together integrate actin and MT polymer regulation to govern critical cellular processes.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Bruce L. Goode