Biochemical and Cellular Imaging Studies of a Novel CDC42-Dependent Formin Pathway

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2006-05-16

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Abstract

The Rho GTPases are important regulators of actin cytoskeletal dynamics during processes such as cell migration, cell polarization and cell division. Different Rho family members exert their effects on actin through specific downstream effectors including members of the WASP and Diaphanous-Related Formin (DRF) protein families. It is presently unclear if, and by what mechanisms, the level, timing and localization of Rho GTPase activity control and coordinate effector activity to produce different types of cytoskeletal structures and rearrangements. On a molecular level, autoinhibition is a common regulatory mechanism for many Rho GTPase effectors. Relief of autoinhibition of WASP by the Rho family member Cdc42 involves a significant GTPase-induced conformational change. Based on this conformational change, I have created a series of single-molecule, FRET-based sensors for active Cdc42 that can faithfully report on Cdc42 activity in vitro and in cells. These sensors may be valuable tools for studying the spatio-temporal dynamics of Cdc42 signaling in vivo. The mechanisms of autoinhibition and activation are less well understood for the DRF family of GTPase effectors. DRFs are characterized by a C-terminal Diaphanous Autoregulatory Domain (DAD) that is postulated to regulate the actin assembly activity of the adjacent formin homology 2 (FH2) domain through autoinhibitory interactions with an N-terminal regulatory region, although this has only been shown directly for the DRF mDia1. Here, I show that the actin assembly activity of FRLα, a macrophage-specific DRF, is also autoinhibited by its N-terminal domain. In cells, autoinhibitory interactions also block a novel GTPase-independent membrane localization activity of the N-terminal domain in both FRLα and mDia1. Autoinhibitory control of FRLα activity and localization are specifically relieved by Cdc42. Timelapse microscopy was used to address the potential physiological significance of the Cdc42-FRLα interaction during Fc-γ receptor mediated phagocytosis in macrophages, a Cdc42-dependent process. The data show that FRLα is required for efficient Fc-γ receptor mediated phagocytosis and that it is recruited to the phagocytic cup by Cdc42. These results suggest mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate an important role for a novel Cdc42-formin pathway in immune function.

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The file named "sethabhinav.pdf" is the primary dissertation file. Supplemental videos (Movies 1-5) are also provided, and more information about each video is available in the file named "Legend.pdf".

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