Abstract
Formins are a conserved class of proteins expressed in all eukaryotes, with known roles in generating cellular actin-based structures. The mammalian formin, FRLalpha, is enriched in hematopoietic cells and tissues, but its biochemical properties have not been characterized. We show that a construct composed of the C-terminal half of FRLalpha (FRLalpha-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing. These multiple activities can be explained by a model in which FRLalpha-C binds filament sides but prefers the topology of sides at the barbed end (end-sides) to those within the filament. This preference allows FRLalpha-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend. Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding model for formins. Profilin partially relieves barbed end elongation inhibition by FRLalpha-C. When non-muscle actin is used, FRLalpha-C's effects are largely similar. FRLalpha-C's ability to sever filaments is the first such activity reported for any formin. Because we find that mDia1-C does not sever efficiently, severing may not be a property of all formins.
Highlights
Budding yeast formins Bni1p and Bnr1p are required for the assembly of actin cables and cytokinetic actin rings in vivo [6, 7]
We show that a construct composed of the C-terminal half of FRL␣ (FRL␣-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing
These multiple activities can be explained by a model in which FRL␣-C binds filament sides but prefers the topology of sides at the barbed end to those within the filament. This preference allows FRL␣-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend. Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding complex is a well characterized nucleation factor, forming networks of branched actin filaments that are present in lamellipodia and ruffles [2]
Summary
Arp2/3, actin-related protein 2/3 complex; BSA, bovine serum albumin; DTT, dithiothreitol; FH1 and FH2, malian formin, FRL (formin-related gene in leukocytes), first identified from mouse spleen as the 1094-amino acid FRL␣ splice variant [15], a number of other variants exist in the data base. Formin homology domains 1 and 2; FRL, formin-related gene in leukocytes; GST, glutathione S-transferase; mDia, mammalian Diaphanous formin; NEM, N-ethylmaleimide. FRL␣-C slows barbed end elongation with an IC50 of about 2 nM, demonstrating that it binds preferentially to filament barbed ends. Additional experiments with platelet actin demonstrate that FRL␣-C has similar effects on non-muscle actin. We postulate that FRL␣-C’s multiple effects on actin dynamics are due to its ability to interact with filament sides, with a preference for the side of the barbed end
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