![]() Despite our familiarity with many of the key regulatory proteins involved in this process, our understanding of the mode by which these proteins cooperate and the sequential manner in which they function remains limited. A variety of small GTP-binding proteins regulate receptor recycling. Once internalized, receptors can either be degraded or recycled back to the plasma membrane. n > 120 per treatment t-test P 21 per treatment t-test P 40 growth cones per genotype t-test *** P < 0.0001 data shown, mean ± s.e.m.Endocytosis is a conserved process across species in which cell surface receptors and lipids are internalized from the plasma membrane. a.u., arbitrary units.Ī, Negative-staining electron microscopy shows that Mical redoxCH + NADPH significantly decreases F-actin length. C, Pyrene-actin assays, where the fluorescence of polymerized pyrene-actin is higher than monomeric pyrene-actin, reveal that purified Mical redoxCH + NADPH directly alters actin polymerization ( a) and induces depolymerization ( b), as do high-speed sedimentation/Coomassie staining assays ( c arrow). n ≥ 2 per condition data shown, mean ± s.e.m. Arrowheads, Mical redoxCH dots, Nus MT, microtubules BSA, bovine serum albumin PHBH, p-hydroxybenzoate hydroxylase MAPs, microtubule-associated proteins Sol, G-actin (soluble) Pel, F-actin (pellet). Purified Mical robustly and selectively associates with F-actin as revealed by dot-blot ( b) and actin and microtubule co-sedimentation/pelleting ( c, d) assays. B, Mical co-localizes (yellow) with F-actin during early and late (inset) stages of bristle elongation ( a). n ≥ 28 bristles per genotype t-test ** P < 0.001, *** P < 0.0001 data shown, mean ± s.e.m. PIR, plexin-interacting region cyto, cytoplasmic portion. Mical therefore is a novel F-actin-disassembly factor that provides a molecular conduit through which actin reorganization-a hallmark of cell morphological changes including axon navigation-can be precisely achieved spatiotemporally in response to semaphorins.Ī, Mical ( GFPMical) co-localizes (yellow) with plexA at sites of bristle branch formation (arrowhead and inset at a ×2 magnified view) and is activated and required for semaphorin–plexin-dependent branching. We also found that Mical utilizes its redox activity to alter F-actin dynamics in vivo and in vitro, indicating a previously unknown role for specific redox signalling events in actin cytoskeletal regulation. Likewise, we purified Mical protein and found that it directly binds F-actin and disassembles both individual and bundled actin filaments. We found that Mical is both necessary and sufficient for semaphorin-plexin-mediated F-actin reorganization in vivo. Here we report that Mical directly links semaphorins and their plexin receptors to the precise control of actin filament (F-actin) dynamics. However, nothing is known of the role of Mical or its redox activity in mediating morphological changes. ![]() Mical enzymes perform reduction-oxidation (redox) enzymatic reactions and also contain domains found in proteins that regulate cell morphology. Recently, highly unusual proteins of the Mical family of enzymes have been found to associate with the cytoplasmic portion of plexins, which are large cell-surface semaphorin receptors, and to mediate axon guidance, synaptogenesis, dendritic pruning and other cell morphological changes. Semaphorins/collapsins were characterized in part on the basis of their ability to drastically alter actin cytoskeletal dynamics in neuronal processes, but despite considerable progress in the identification of semaphorin receptors and their signalling pathways, the molecules linking them to the precise control of cytoskeletal elements remain unknown. Semaphorins are one of the largest families of these instructive cues and are widely studied for their effects on cell movement, navigation, angiogenesis, immunology and cancer. How instructive cues present on the cell surface have their precise effects on the actin cytoskeleton is poorly understood.
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