Disassembly Of Actin Filaments Research Articles

Fascin-induced bundling protects actin filaments from disassembly by cofilin.

Actin filament turnover plays a central role in shaping actin networks, yet the feedback mechanism between network architecture and filament assembly dynamics remains unclear. The activity of ADF/cofilin, the main protein family responsible for filament disassembly, has been mainly studied at the single filament level. This study unveils that fascin, by crosslinking filaments into bundles, strongly slows down filament disassembly by cofilin. We show that this is due to a markedly slower initiation of the first cofilin clusters, which occurs up to 100-fold slower on large bundles compared with single filaments. In contrast, severing at cofilin cluster boundaries is unaffected by fascin bundling. After the formation of an initial cofilin cluster on a filament within a bundle, we observed the local removal of fascin. Notably, the formation of cofilin clusters on adjacent filaments is highly enhanced, locally. We propose that this interfilament cooperativity arises from the local propagation of the cofilin-induced change in helicity from one filament to the other filaments of the bundle. Overall, taking into account all the above reactions, we reveal that fascin crosslinking slows down the disassembly of actin filaments by cofilin. These findings highlight the important role played by crosslinkers in tuning actin network turnover by modulating the activity of other regulatory proteins.

Differential sensitivity of ADF isovariants to a pH gradient promotes pollen tube growth.

The actin cytoskeleton is one of the targets of the pH gradient in tip-growing cells, but how cytosolic pH regulates the actin cytoskeleton remains largely unknown. We here demonstrate that Arabidopsis ADF7 and ADF10 function optimally at different pH levels when disassembling actin filaments. This differential pH sensitivity allows ADF7 and ADF10 to respond to the cytosolic pH gradient to regulate actin dynamics in pollen tubes. ADF7 is an unusual actin-depolymerizing factor with a low optimum pH in in vitro actin depolymerization assays. ADF7 plays a dominant role in promoting actin turnover at the pollen tube apex. ADF10 has a typically high optimum pH in in vitro assays and plays a dominant role in regulating the turnover and organization of subapical actin filaments. Thus, functional specification and cooperation of ADF isovariants with different pH sensitivities enable the coordination of the actin cytoskeleton with the cytosolic pH gradient to support pollen tube growth.

Modification of phosphoinositides by the Shigella effector IpgD during host cell infection.

Shigella, the causative agent of bacillary dysentery, subvert cytoskeletal and trafficking processes to invade and replicate in epithelial cells using an arsenal of bacterial effectors translocated through a type III secretion system. Here, we review the various roles of the type III effector IpgD, initially characterized as phosphatidylinositol 4,5 bisphosphate (PI4,5P2) 4-phosphatase. By decreasing PI4,5P2 levels, IpgD triggers the disassembly of cortical actin filaments required for bacterial invasion and cell migration. PI5P produced by IpgD further stimulates signaling pathways regulating cell survival, macropinosome formation, endosomal trafficking and dampening of immune responses. Recently, IpgD was also found to exhibit phosphotransferase activity leading to PI3,4P2 synthesis adding a new flavor to this multipotent bacterial enzyme. The substrate of IpgD, PI4,5P2 is also the main substrate hydrolyzed by endogenous phospholipases C to produce inositoltriphosphate (InsP3), a major Ca2+ second messenger. Hence, beyond the repertoire of effects associated with the direct diversion of phoshoinositides, IpgD indirectly down-regulates InsP3-mediated Ca2+ release by limiting InsP3 production. Furthermore, IpgD controls the intracellular lifestyle of Shigella promoting Rab8/11 -dependent recruitment of the exocyst at macropinosomes to remove damaged vacuolar membrane remnants and promote bacterial cytosolic escape. IpgD thus emerges as a key bacterial effector for the remodeling of host cell membranes.

MICAL1 activation by PAK1 mediates actin filament disassembly.

The MICAL1 monooxygenase is an important regulator of filamentous actin (F-actin) structures. Although MICAL1 has been shown to be regulated via protein-protein interactions at the autoinhibitory carboxyl terminus, a link between actin-regulatory RHO GTPase signaling pathways and MICAL1 has not been established. We show that the CDC42 GTPase effector PAK1 associates with and phosphorylates MICAL1 on two serine residues, leading to accelerated F-actin disassembly. PAK1 binds to the amino-terminal catalytic monooxygenase and calponin homology domains, distinct from the autoinhibitory carboxyl terminus. Extracellular ligand stimulation leads to PAK-dependent phosphorylation, linking external signals to MICAL1 phosphorylation. Mass spectrometry indicates that MICAL1 co-expression with CDC42 and PAK1 increases MICAL1 association with hundreds of proteins, including the previously described MICAL1-interacting proteins RAB10 and RAB7A. These results provide insights into a redox-mediated pathway linking extracellular signals to cytoskeleton regulation via a RHO GTPase and indicate a means of communication between RHO and RAB GTPases.

F-actin dynamics in midgut cells enables virus persistence in vector insects.

Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects.

Biochemical and mechanical regulation of actin dynamics.

Polymerization of actin filaments against membranes produces force for numerous cellular processes, such as migration, morphogenesis, endocytosis, phagocytosis and organelle dynamics. Consequently, aberrant actin cytoskeleton dynamics are linked to various diseases, including cancer, as well as immunological and neurological disorders. Understanding how actin filaments generate forces in cells, how force production is regulated by the interplay between actin-binding proteins and how the actin-regulatory machinery responds to mechanical load are at the heart of many cellular, developmental and pathological processes. During the past few years, our understanding of the mechanisms controlling actin filament assembly and disassembly has evolved substantially. It has also become evident that the activities of key actin-binding proteins are not regulated solely by biochemical signalling pathways, as mechanical regulation is critical for these proteins. Indeed, the architecture and dynamics of the actin cytoskeleton are directly tuned by mechanical load. Here we discuss the general mechanisms by which key actin regulators, often in synergy with each other, control actin filament assembly, disassembly, and monomer recycling. By using an updated view of actin dynamics as a framework, we discuss how the mechanics and geometry of actin networks control actin-binding proteins, and how this translates into force production in endocytosis and mesenchymal cell migration.

Oxidation and reduction of actin: Origin, impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today's availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

Magic angle spinning NMR structure of human cofilin-2 assembled on actin filaments reveals isoform-specific conformation and binding mode

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly.

Dok-1 regulates mast cell degranulation negatively through inhibiting calcium-dependent F-actin disassembly

In food allergies, antigen-induced aggregation of FcεRI on mast cells initiates highly ordered and sequential signaling events. Dok-1(downstream of tyrosine kinase 1), undergoes intense tyrosine phosphorylation upon FcεRI stimulation, which negatively regulates Ras/Erk signaling and the subsequent cytokine release, but it remains unclear whether Dok-1 regulates Fc-mediated degranulation. In this study, we investigated the role of Dok-1 in FcεRI-mediated degranulation. Dok-1 overexpressing RBL-2H3 cells were established. Degranulation, immunoprecipitation, co-immunoprecipitation, immunoblotting and flow cytometry assay were performed to explore the effects of Dok-1 and its underlying mechanisms. We found that, following FcεRI activation, Dok-1 was recruited to the plasma membrane, leading to tyrosine phosphorylation. Phosphorylated Dok-1 inhibits FcεRI-operated calcium influx, and negatively regulated degranulation by inhibiting calcium-dependent disassembly of actin filaments. Our data revealed that Dok-1 is a negative regulator of FcεRI-mediated mast cell degranulation. These findings contribute to the identification of therapeutic targets for food allergies.

Celebrating 20 years of live single-actin-filament studies with five golden rules

The precise assembly and disassembly of actin filaments is required for several cellular processes, and their regulation has been scrutinized for decades. Twenty years ago, a handful of studies marked the advent of a new type of experiment to study actin dynamics: using optical microscopy to look at individual events, taking place on individual filaments in real time. Here, we summarize the main characteristics of this approach and how it has changed our ability to understand actin assembly dynamics. We also highlight some of its caveats and reflect on what we have learned over the past 20 years, leading us to propose a set of guidelines, which we hope will contribute to a better exploitation of this powerful tool.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5–primed eosinophils causes cell death via CD11b/CD18 integrin–mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8–induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8–induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5–primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Mapping the Proximity Interaction Network of STIM1 Reveals New Mechanisms of Cytoskeletal Regulation.

Stromal interaction molecule 1 (STIM1) resides primarily in the sarco/endoplasmic reticulum, where it senses intraluminal Ca2+ levels and activates Orai channels on the plasma membrane to initiate Ca2+ influx. We have previously shown that STIM1 is involved in the dynamic remodeling of the actin cytoskeleton. However, the downstream effectors of STIM1 that lead to cytoskeletal remodeling are not known. The proximity-labeling technique (BioID) can capture weak and transient protein-protein interactions, including proteins that reside in the close vicinity of the bait, but that may not be direct binders. Hence, in the present study, we investigated the STIM1 interactome using the BioID technique. A promiscuous biotin ligase was fused to the cytoplasmic C-terminus of STIM1 and was stably expressed in a mouse embryonic fibroblast (MEF) cell line. Screening of biotinylated proteins identified several high confidence targets. Here, we report Gelsolin (GSN) as a new member of the STIM1 interactome. GSN is a Ca2+-dependent actin-severing protein that promotes actin filament assembly and disassembly. Results were validated using knockdown approaches and immunostaining. We tested our results in neonatal cardiomyocytes where STIM1 overexpression induced altered actin dynamics and cytoskeletal instability. This is the first time that BioID assay was used to investigate the STIM1 interactome. Our work highlights the role of STIM1/GSN in the structure and function of the cytoskeleton.

Functional Redundancy of Cyclase-Associated Proteins CAP1 and CAP2 in Differentiating Neurons

Cyclase-associated proteins (CAPs) are evolutionary-conserved actin-binding proteins with crucial functions in regulating actin dynamics, the spatiotemporally controlled assembly and disassembly of actin filaments (F-actin). Mammals possess two family members (CAP1 and CAP2) with different expression patterns. Unlike most other tissues, both CAPs are expressed in the brain and present in hippocampal neurons. We recently reported crucial roles for CAP1 in growth cone function, neuron differentiation, and neuron connectivity in the mouse brain. Instead, CAP2 controls dendritic spine morphology and synaptic plasticity, and its dysregulation contributes to Alzheimer’s disease pathology. These findings are in line with a model in which CAP1 controls important aspects during neuron differentiation, while CAP2 is relevant in differentiated neurons. We here report CAP2 expression during neuron differentiation and its enrichment in growth cones. We therefore hypothesized that CAP2 is relevant not only in excitatory synapses, but also in differentiating neurons. However, CAP2 inactivation neither impaired growth cone morphology and motility nor neuron differentiation. Moreover, CAP2 mutant mice did not display any obvious changes in brain anatomy. Hence, differently from CAP1, CAP2 was dispensable for neuron differentiation and brain development. Interestingly, overexpression of CAP2 rescued not only growth cone size in CAP1-deficient neurons, but also their morphology and differentiation. Our data provide evidence for functional redundancy of CAP1 and CAP2 in differentiating neurons, and they suggest compensatory mechanisms in single mutant neurons.

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM-based single-cell force spectroscopy

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. In-vitro single molecule force spectroscopy experiments can reveal cadherin–cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin-presenting cell homophilic and heterophilic binding in the proteins’ native conformational and functional states in living cells has rarely been done. Here, we used atomic force microscopy (AFM) based single-cell force spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N- (neural), OB- (osteoblast) and E- (epithelial) cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e., between cells and cadherins of the same and different types. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E-cadherin homophilic binding events are stronger than N/OB heterophilic binding events. Disassembly of intracellular actin filaments affects the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements, but particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins’ homophilic bonds.

Cytoskeletal Dynamics in Epithelial-Mesenchymal Transition: Insights into Therapeutic Targets for Cancer Metastasis.

Simple SummaryThe epithelial to mesenchymal transition (EMT) is a well-documented process in the study of cancer metastases. The cytoskeleton is an intricate network involved in various cellular activities and impacts cell shape, division, trafficking, and motility. However, several functions and activities of the cytoskeleton, which plays a pivotal role in EMT, are not fully understood. This review aims to provide significant insights into the cytoskeleton’s physiological functions and the crucial role in the EMT process. Our review focuses on the participation of actin filaments, intermediate filaments, and microtubules in promoting EMT and their influence on cancer metastasis. We have also highlighted potential therapeutic targets associated with EMT activation for clinical intervention. A better understanding of multi-drug resistance (MDR) mechanisms in cancer cells with the cytoskeleton could accelerate the discovery of new therapies for aggressive cancer.In cancer cells, a vital cellular process during metastasis is the transformation of epithelial cells towards motile mesenchymal cells called the epithelial to mesenchymal transition (EMT). The cytoskeleton is an active network of three intracellular filaments: actin cytoskeleton, microtubules, and intermediate filaments. These filaments play a central role in the structural design and cell behavior and are necessary for EMT. During EMT, epithelial cells undergo a cellular transformation as manifested by cell elongation, migration, and invasion, coordinated by actin cytoskeleton reorganization. The actin cytoskeleton is an extremely dynamic structure, controlled by a balance of assembly and disassembly of actin filaments. Actin-binding proteins regulate the process of actin polymerization and depolymerization. Microtubule reorganization also plays an important role in cell migration and polarization. Intermediate filaments are rearranged, switching to a vimentin-rich network, and this protein is used as a marker for a mesenchymal cell. Hence, targeting EMT by regulating the activities of their key components may be a potential solution to metastasis. This review summarizes the research done on the physiological functions of the cytoskeleton, its role in the EMT process, and its effect on multidrug-resistant (MDR) cancer cells—highlight some future perspectives in cancer therapy by targeting cytoskeleton.

Cyclase-associated protein 2 (CAP2) controls MRTF-A localization and SRF activity in mouse embryonic fibroblasts

Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity in CAP2 mutant MEFs was associated with altered actin turnover, a shift in the actin equilibrium towards monomeric actin (G-actin) as well as and reduced nuclear levels of myocardin-related transcription factor A (MRTF-A), a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation with jasplakinolide restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.

Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion.

Cell-cell fusion is a frequent and essential event during development, and its dysregulation causes diseases ranging from infertility to muscle weakness. Fusing cells need to repeatedly remodel their plasma membrane through orchestrated formation and disassembly of actin filaments, but how the dynamic reorganization of the cortical actin cytoskeleton is controlled is still poorly understood. Here, we identified a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. We found that EPS8-IRSp53 complexes stabilize cortical actin bundles at sites of cell contact to promote close membrane alignment. EPS8 monoubiquitylation by CUL3KCTD10 displaces EPS8-IRSp53 from membranes and counteracts actin bundling, a dual activity that restricts actin bundling to allow paired cells to progress with fusion. We conclude that cytoskeletal rearrangements during development are precisely controlled by ubiquitylation, raising the possibility of modulating the efficiency of cell-cell fusion for therapeutic benefit.

Actin Turnover Regulation through Myosin Activity and Network Architecture

Actin remodeling through turnover is essential for the survival of most cells. However, the regulation of this process through actin architecture and myosin activity is poorly studied. While myosin-mediated disassembly of actin filaments has been implicated in regulating turnover in cells (Wilson et al. Nature 2010), the underlying dynamics of this process, and the effects of actin structure, have not been studied. Here, we investigate the ability of myosin activity and actin architecture to control turnover. Using a minimal system to recreate turnover in vitro, we observe that severing by cofilin increases the turnover rate in our networks. We additionally find that myosin mediated severing is sufficient to increase the rate of actin turnover, even in systems without cofilin. Preventing filament depolymerization through the addition of phalloidin or reducing myosin-mediated buckling by shortening the length of filaments reduces this effect, suggesting that the increased rate relies on actin severing and disassembly. This increase in turnover, however, is dependent on actin structure. When actin is bundled by the cross-linker α-actinin, cofilin mediated turnover vanishes. Remarkably, α-actinin does not impact myosin mediated severing, and myosin is able to increase turnover even in bundled networks. These results suggest that not only can myosin regulate turnover of actin filaments, but also that different methods of disassembly might be needed to remodel actin depending on its local structure.

Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly.

Proteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non-activated, phosphomimetic S3D-cofilin binds and severs oxidized actin filaments rapidly, in conditions where non-oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1-induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that, in cells, direct post-translational modification of actin filaments by oxidation is a way to trigger their disassembly.