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

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Title: Biochemical and Cellular Imaging Studies of a Novel CDC42-Dependent Formin Pathway
Author: Seth, Abhinav
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 singlemolecule , 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 FRLa , 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 FRLa and mDia1 . Autoinhibitory control of FRLa activity and localization are specifically relieved by Cdc42 . Timelapse microscopy was used to address the potential physiological significance of the Cdc42 -FRLa interaction during Fc -gamma receptor mediated phagocytosis in macrophages , a Cdc42 -dependent process . The data show that FRLa is required for efficient Fc -gamma 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 .
URI: http : / /hdl .handle .net /2152 .5 /347
Date: 2006-05-16


Biochemical and Cellular Imaging Studies of a Novel CDC42-Dependent Formin Pathway. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /347 .

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