Research Project
10217192
Signaling from membranes to the actin cytoskeleton underpins a myriad of processes, such as cell migration and vesicle trafficking, and its dysregulation leads to various diseases, including cancer and neurological dis- orders. The WAVE Regulatory Complex (WRC) of ~400 kDa plays a central role in linking membrane signals to the actin cytoskeleton throughout biology. The WRC alone is autoinhibited, but a large variety of membrane ligands can activate the complex through direct interactions, which, in turn, stimulates the Arp2/3 complex to polymerize actin. These ligands include the GTPases Rac1 and Arf1, and over 100 different membrane pro- teins previously identified by the applicant. Despite this long list of WRC ligands and their broad biological func- tions, it remains unknown how these membrane molecules, individually or cooperatively, control WRC?s activity and thereby actin assembly. One major challenge is to biochemically reconstitute the multivalent, weak interac- tions of the WRC with its ligands, especially with its canonical activators Rac1 and Arf1. Preliminary data demonstrate that it is now possible to reconstitute stable WRC/ligand complexes by using recombinant materi- al and by different tethering strategies. The unique access to such material will enable determination of the bi- ochemical and structural mechanisms mediating WRC activation. Preliminary data suggest that activation of the WRC requires simultaneous binding of two Rac1 molecules at two distinct sites and that the activation is enhanced by a third interaction with Arf1. Other membrane ligands may further modulate the activation by in- teractions at additional, distinct sites. My lab will target three major aspects of WRC activation by biochemical and structural approaches. Project 1 will determine the structure of the WRC simultaneously bound to two Rac1 molecules. This will reveal the first activated structure of the WRC and will provide a mechanistic frame- work for understanding WRC activation. Project 2 will combine biochemical and structural approaches to define how the WRC is activated by Arf1 and how Arf1 and Rac1 act cooperatively to optimize activation. This will re- veal new mechanisms of WRC activation and open new avenues for understanding actin regulation in many processes controlled by the Arf family of GTPases. Project 3 will discover novel interaction mechanisms of the WRC with three newly identified membrane ligands, including the claudin-like receptor HPO-30, the neuronal receptor Retrolinkin, and the kainate family of glutamate receptors. This will reveal new modes of actin regula- tion mediated by membrane receptors and the WRC. Together, by generating previously unattainable material and by closely integrating quantitative biochemistry and structural approaches, successful completion of this work will provide a comprehensive, unifying framework for understanding WRC activation, knowledge that will be broadly applicable to many different cellular systems and biological processes widely regulated by this sig- naling hub and its many ligands. Our work will also provide new structural targets for therapeutics and will have a broad impact in areas ranging from cell biology and immunology to neuroscience and developmental biology.
