Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF/cofilins/twinstar, sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared to either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodeling, axon guidance, and Semaphorin/Plexin repulsion. Mical and cofilin, therefore, form a Redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties.

Cellular events require the spatiotemporal interplay between actin assembly and actin disassembly. Yet, how different factors promote the integration of these two opposing processes is unclear. In particular, cellular monomeric (G)-actin is complexed with profilin, which inhibits spontaneous actin nucleation but fuels actin filament (F-actin) assembly by elongation-promoting factors (formins, Ena/VASP). In contrast, site-specific F-actin oxidation by Mical promotes F-actin disassembly and release of polymerization-impaired Mical-oxidized (Mox)-G-actin. Here we find that these two opposing processes connect with one another to orchestrate actin/cellular remodeling. Specifically, we find that profilin binds Mox-G-actin, yet these complexes do not fuel elongation factors’-mediated F-actin assembly, but instead inhibit polymerization and promote further Mox-F-actin disassembly. Using Drosophila as a model system, we show that similar profilin–Mical connections occur in vivo – where they underlie F-actin/cellular remodeling that accompanies Semaphorin–Plexin cellular/axon repulsion. Thus, profilin and Mical combine to impair F-actin assembly and promote F-actin disassembly, while concomitantly facilitating cellular remodeling and plasticity.

The F-actin cytoskeleton drives cellular form and function. However, how F-actin-based changes occur with spatiotemporal precision and specific directional orientation is poorly understood. Here, we identify that the unconventional class XV myosin [Myosin 15 (Myo15)] physically and functionally interacts with the F-actin disassembly enzyme Mical to spatiotemporally position cellular breakdown and reconstruction. Specifically, while unconventional myosins have been associated with transporting cargo along F-actin to spatially target cytoskeletal assembly, we now find they also target disassembly. Myo15 specifically positions this F-actin disassembly by associating with Mical and using its motor and MyTH4-FERM cargo-transporting functions to broaden Mical’s distribution. Myo15’s broadening of Mical’s distribution also expands and directionally orients Mical-mediated F-actin disassembly and subsequent cellular remodeling, including in response to Semaphorin/Plexin cell surface activation signals. Thus, we identify a mechanism that spatiotemporally propagates F-actin disassembly while also proposing that other F-actin-trafficked-cargo is derailed by this disassembly to directionally orient rebuilding.

Understanding how neurons form, extend, and navigate their finger-like axonal and dendritic processes is crucial for developing therapeutics for the diseased and damaged brain. Although less well appreciated, many other types of cells also send out similar finger-like projections. Indeed, unlike neuronal specific phenomena such as synapse formation or synaptic transmission, an important issue for thought is that this critical long-standing question of how a cellular process like an axon or dendrite forms and extends is not primarily a neuroscience problem but a cell biological problem. In that case, the use of simple cellular processes – such as the bristle cell process of Drosophila – can aid in the fight to answer these critical questions. Specifically, determining how a model cellular process is generated can provide a framework for manipulations of all types of membranous process-containing cells, including different types of neurons.

cell membrane and accumulate F-to toxic levels. One mechanism developed to mitigate this problem is the Fluc family of fluoride channels. These channels, extremely selective for fluoride over chloride and other anions, allow passive draining of fluoride down to sub-toxic levels. Recent structural work on two homologues of these proteins has demonstrated some very unusual characteristics. Like the small multidrug resistance transporter EmrE, Fluc is a dual-topology membrane protein, where the functional channel is an antiparallel homodimer made up of two Fluc molecules inserted into the membrane in opposite orientations. Surprisingly, structures of the Fluc proteins do not show a single, clear pore through the center of the dimer. Rather, there appears to be two independent ion pathways through the channel, with residues from both protein chains contributing to both pathways. We are currently using a combination of mutagenesis, electrophysiology, and crystallography to attempt to clearly delineate the pathways of ion conduction through the channel, and try to determine conclusively whether Fluc has a ''double-barreled'', parallel channel architecture. To that end, we have produced a functional Fluc concatomer, with both members of the antiparallel dimer contained in a single protein chain, linked by an additional transmembrane helix to maintain the antiparallel structure. This construct has allowed us to mutagenically degrade each pore independently, and thus demonstrate the presence of two independent pores through this small channel. 1747-PlatVoltage Sensitivity of the Bacterial Protein Translocation Channel The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes.The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.