Multicomponent Uncapping of Actin Filaments by Twinfilin and Formin Pubblico
Reddy, Vishal (Spring 2024)
Abstract
Multicomponent Uncapping of Actin Filaments by Twinfilin and Formin
The actin cytoskeleton plays a major role in determining cell shape, polarity, and flexibility as well as driving various cellular processes. Capping Protein (CP) arrests growing actin filament barbed ends by halting both polymerization and depolymerization. However, until CP is removed, the barbed end cannot be accessed by other actin-binding proteins, thus the intermediate step of uncapping must occur to dissociate CP. Though this process occurs rapidly in cells, laboratory experiments have revealed a relatively longer dwell time for CP on barbed ends. The disparity between observed rates in vivo and in vitro is thought to be due to the action of a class of actin-binding proteins known as uncappers. The effects of twinfilin (mTwinfilin-1) and formin (mDia1) on uncapping rates was tested using microfluidics-assisted Total Internal Reflection Microscopy (mf-TIRF). The effects of filament nucleotide state, a commonly understood influencer of actin filament biochemistry, were also accounted for. We found that twinfilin increased uncapping rates of both ADP and ADP-Pi state filaments by 8-fold and 3-fold respectively, saturating at a concentration of 5 µM twinfilin. We observed formin’s effects on ADP and ADP-Pi filaments uncapping as a linear increase in uncapping rate with a maximum observed increase of 37-fold and 5-fold respectively at 200 nM formin. ADP and ADP-Pi filaments also appeared to possess different CP dissociation rates in the absence of other proteins as well, with ADP filaments generally dissociating CP 3-fold faster than ADP-Pi. When present together, twinfilin and formin displayed a synergistic effect on uncapping rates for ADP and ADP-Pi filaments, and we observed a rate increase by about 270-fold and 8-fold respectively, for 5 µM twinfilin and 200 nM formin. Taken together, these results suggest that actin filament barbed ends in the ADP state are more prone to uncapping than barbed ends in the ADP-Pi state due to a combination of weaker CP binding and increased susceptibility to the action of uncappers such as twinfilin and formin.
Table of Contents
Chapter 1 Introduction pp. 1-4
Chapter 2 Experimental Techniques pp. 5-8
Chapter 3 Results pp. 9-12
Chapter 4 Discussion pp. 13-14
Chapter 5 Future Directions pp. 15
Figures pp. 16-28
References pp. 29-32
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