Abstract
Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP–G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of “muscle” (CFL2) and “non-muscle” (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.
Highlights
A dynamic balance between the polymerization of actin filaments at the barbed ends and depolymerization at the pointed ends is crucial for numerous cellular processes including cell adhesion, cell motility, cytokinesis, and morphogenesis [1,2,3,4]
helical folded domain (HFD) interacts with F-actin and enhances cofilin-mediated F-actin disassembly [31,38], while the coiled coil contributes to oligomerization [38]
We employed sedimentation velocity analytical ultracentrifugation (SV-AUC) to determine the oligomerization states of the recombinant constructs of human CAP1 and CAP2 HFDs with the original N-terminal coiled coils (N-CAP1 and N-CAP2) and 6xHis-tags placed at the C-termini to avoid interference with oligomerization
Summary
A dynamic balance between the polymerization of actin filaments at the barbed ends and depolymerization at the pointed ends is crucial for numerous cellular processes including cell adhesion, cell motility, cytokinesis, and morphogenesis [1,2,3,4]. This issue was resolved, by the discovery of proteins that assist ADF/cofilins in the controlled acceleration of actin depolymerization: actin-interacting protein 1 (Aip1), coronin, twinfilin, and cyclase-associated protein (Srv2/CAP) [22,23,24,25,26] Working together, these proteins enable the mechanisms of selective nucleotide-dependent depolymerization of aged filaments [25,27], prevent polymerization at the barbed ends formed as a result of severing [26], accelerate dissociation of actin subunits from severed filaments [24], and stimulate dissociation of ADP from the depolymerized G-actin and its recharging by ATP [28]. The newly formed ATP–G-actin monomer is either released or actively transferred to profilin, completing the goal of regenerating the pool of polymerization-competent actin [28,35,36,37]
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