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

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dc.contributor.advisor Rosen , Michael K . en
dc.creator Seth , Abhinav en
dc.date.accessioned 2010 -07 -12T17 :42 :08Z en
dc.date.accessioned 2014 -02 -19T22 :01 :59Z
dc.date.available 2010 -07 -12T17 :42 :08Z en
dc.date.available 2014 -02 -19T22 :01 :59Z
dc.date.issued 2006 -05 -16 en
dc.identifier.other en
dc.identifier.uri http : / /hdl .handle .net /2152 .5 /347 en
dc.description.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 . en
dc.format.medium Electronic en
dc.format.mimetype application /pdf en
dc.language.iso en en
dc.subject Actins en
dc.subject rho GTP -Binding Proteins en
dc.subject Cell Physiological Processes en
dc.title Biochemical and Cellular Imaging Studies of a Novel CDC42 -Dependent Formin Pathway en
dc.type.genre dissertation en
dc.type.material Text en
thesis.degree.name Doctor of Philosophy en
thesis.degree.level Ph .D . en
thesis.degree.discipline Cell Regulation en
thesis.degree.grantor Graduate School of Biomedical Sciences en
thesis.degree.department en
dc.format.digitalOrigin born digital en
thesis.date.available 2006 -05 -16 en

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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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