We found that elongation of Mox-actin is ~3 fold slower than that of control actin under the same conditions (Fig. To characterize the dynamic properties of purified Mical-oxidized (Mox) actin, we employed single filament total internal reflection microscopy (TIRFM). Nucleotide-state dependent instability of Mical-oxidized actin Thus, Mical-induced oxidation of actin-including augmentation of cofilin severing-provides a robust mechanism to disassemble different actin forms (ATP/ADP-Pi- and ADP-bound) in response to cellular signaling. Moreover, we show that oxidation by Mical makes phosphate-rich (“young”) actin susceptible to cofilin severing. Site-directed mutagenesis indicated that this nascent interaction weakens protomer-protomer contacts to facilitate catastrophic F-actin disassembly. One of these structural conformers suggested a new intermolecular interaction that occurs upon Mical oxidation of the actin residue M47 (M47-O-T351). In agreement with our TIRFM data, atomic modeling based on the 3.9 Å resolution cryoEM structure of Mox-F-actin resolved two main structural states of Mical-oxidized filaments.
We show that one of these states undergoes extremely rapid (catastrophic) disassembly (84 subunits/s) in a phosphate/BeFx sensitive manner. Here, we identify two dynamic states of Mox-F-actin using single filament TIRFM. However, the molecular basis of such dynamic behavior of Mical-oxidized (Mox) actin and the mechanisms underlying its disassembly are hitherto unknown. Most recently, rapid depolymerization of actin filaments upon Mical/NADPH treatment was observed in in vitro assays 11. F-actin disassembly can be broadly defined as loss of polymer mass due to depolymerization (monomers’ dissociation from filament ends), which is often facilitated by severing (filaments’ fragmentation resulting in increased number of depolymerizing ends). Mical stereo-specifically oxidizes F-actin on methionine (M) 44/47 which induces F-actin disassembly 12-most effectively in conjunction with cofilin 13. Selective redox regulation of actin by Mical family enzymes has been found to promote cellular destabilization of F-actin 2 and play important roles in axonal guidance 3, 4, dendritic organization 5, synaptic development 6 and homeostasis 7, heart 8 and muscle 6 development, cell viability 9, exocytosis 10, and cytokinesis 11. Regulation of actin filament dynamics by post-translational modifications is poorly understood compared to that by non-covalent means, through actin-binding proteins 1. Thus, in conjunction with cofilin, Mical oxidation of actin promotes F-actin disassembly independent of the nucleotide-bound state. Moreover, we find that Mical oxidation of actin allows for cofilin-mediated severing even in the presence of inorganic phosphate. Site-directed mutagenesis reveals that this interaction promotes Mox-actin instability. Modeling actin’s D-loop region based on our 3.9 Å cryoEM reconstruction suggests that oxidation by Mical reorients the side chain of M44 and induces a new intermolecular interaction of actin residue M47 (M47-O-T351). Using near-atomic resolution cryoEM reconstruction and single filament TIRF microscopy we identify two dynamic and structural states of Mox-actin. Here we show that Mical-oxidized (Mox) actin can undergo extremely fast (84 subunits/s) disassembly, which depends on F-actin’s nucleotide-bound state. MICAL Redox enzymes are important post-translational effectors of actin that stereo-specifically oxidize actin’s M44 and M47 residues to induce cellular F-actin disassembly. Actin filament assembly and disassembly are vital for cell functions.
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