Barbed end regulation is critical to the formation of the actin-based protrusive structures, lamellipodia and filopodia. In this body of work we have chosen to focus on two main activities, filament termination by the heterodimeric Capping protein (CP) and continued filament elongation by the Ena/VASP family of proteins. We demonstrate that the transition from lamellipodia-to-filopodia is elegantly controlled by the activities of these two proteins whereby depletion of CP via siRNA leads to a hyper-filopodial phenotype but only under conditions where at least single member of the Ena/VASP family of proteins is expressed. In addition, depletion of CP led to diminution of the lamellipodia, concomitant displacement of the Arp2/3 complex, and abrogated rates lamellipodial protrusion. Through rescue experiments we demonstrated that it is the specific depletion of CP that leads these phenotypes.
Furthermore we demonstrated that Ena/VASP proteins function beyond that of an anti-capper and play a key role in filopodia formation through oligomerization and capture of actin barbed ends. In doing so, we also determined that the C-terminal Ena-VASP homology-2 (EVH2) domain is the minimal domain of filiopodia formation. Furthermore, we determined that upon lamellipodial-to-filopodial transition Ena/VASP proteins change kinetics, becoming "static" at filopodia tips. Despite this "static" association with the barbed ends, Ena/VASP still allow for continuous polymerization suggesting insertional polymerization.
Finally, we putatively uncovered new roles for CP and twinfilin. Localization and kinetics indicate that CP has a discrete lamellipodial distribution and short lifetime on actin barbed ends. There is also a discrepancy between CP's distribution and that of the actin barbed ends. Through perturbation of CP localization by cytochalasin D, as well as a RNAi screen we have uncovered a role for twinfilin as a "global capper" terminating the actin barbed ends not actively engaged in protrusion, while CP functions more locally.
|Advisor:||Borisy, Gary G.|
|Commitee:||Bartles, James R., Gelfand, Vladmir I., Goldman, Robert D., McGarry, Thomas J., Taylor, Edwin|
|Department:||Integrated Graduate Program in the Life Sciences|
|School Location:||United States -- Illinois|
|Source:||DAI-B 68/09, Dissertation Abstracts International|
|Keywords:||Actin, Actin protrusive structures, Capping protein, Ena/VASP, Filopodia, Lamellipodia|
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