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[bioRxiv]Direct visualization of how Actin Depolymerizing Factor's filament severing and depolym


A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping Protein (CP) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. While filament fragmentation by ADF/cofilin has long been demonstrated by TIRF, filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. We have also built a conceptually novel assay to directly visualize ADF's effect on a filament population. We demonstrate that ADF's enhanced pointed end depolymerization leads to an increase in polymerizable actin monomers co-existing with filaments, thus promoting faster barbed end growth. We further reveal how ADF-enhanced filament depolymerization synergizes with CP's long predicted “monomer funneling” and leads to skyrocketing of filament growth rates, close to estimated rates in the lamellipodia. The “Funneling model” hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few noncapped filaments transiently grow much faster, an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling by CP at the scale of individual filaments. We believe that these results enlighten our understanding of the turnover of cellular actin networks.

Shashank Shekhar, Marie-France Carlier doi: https://doi.org/10.1101/114199

Link: http://biorxiv.org/content/early/2017/03/06/114199

#03112017 #filament #actin #TIRFmicroscopy #CellularBiophysics

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