Direct Redox Regulation of F-Actin Assembly and Disassembly by MICAL

How guidance cues present outside of cells exert their precise effects on the internal actin cytoskeleton is poorly understood. Such effects are critical for diverse cellular behaviors including polarity, morphology, adhesion, motility, process elongation, navigation, and connectivity. Semaphorins, for example, are one of the largest families of these guidance signals and play critical roles in neurobiology, angiogenesis, immunology, and cancer. One interesting characteristic of the Semaphorins is that they inhibit the movement of cells (and their membranous processes) through their ability to disrupt actin cytoskeletal organization. However, despite considerable progress in the identification of Semaphorin receptors and their signal transduction pathways, the molecules linking them to the precise control of the actin cytoskeleton have remained mysterious. During my graduate studies, I sought to better understand a family of unusual proteins called the MICALs (which includes one Drosophila Mical and three vertebrate MICALs), which associate with the Semaphorin cell-surface receptor Plexin and are important for Semaphorins to exert their effects. Nothing was known, however, regarding the specific role of the MICALs in these Semaphorin-dependent events. Not long after I began my graduate work, my colleagues and I noticed that Mical was necessary for proper actin cytoskeletal organization and sufficient to reorganize the actin cytoskeleton in vivo. Therefore, to better understand the role that Mical plays in actin cytoskeletal rearrangements, I took a biochemical approach, and purified the Mical protein. Utilizing biochemical and imaging approaches with purified proteins, I found that Mical directly binds to actin filaments (F-actin) and is able to induce the rapid disassembly of F-actin. Thus, my results revealed that Mical is a novel F-actin disassembly factor that provides a molecular conduit through which F-actin disassembly can be precisely achieved in response to Semaphorins. So I next wondered how Mical induces F-actin disassembly. Interestingly, the MICALs belong to a class of flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in oxidationreduction (Redox) reactions. Although MICALs have no known substrate/s, my in vivo and in vitro results revealed that Mical employs its Redox region to bind F-actin and disassembles filaments in an NADPH-dependent manner. Moreover, this Mical-treated actin failed to repolymerize even after removal of Mical, indicating that Mical stably modifies actin to alter polymerization. Mass spectrometric analyses revealed that F-actin subunits were directly modified by Mical on their conserved pointed-end that is critical for filament assembly. Specifically, Mical post-translationally oxidized a conserved amino acid (Methionine 44) within a region of actin that is critical for actin-actin contacts, simultaneously severing filaments and decreasing polymerization. Thus, my thesis observations reveal a novel and specific oxidation dependent signaling mechanism that selectively regulates actin dynamics and cellular behaviors.