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.

Structural basis of rapid actin dynamics in the evolutionarily divergent Leishmania parasite.

Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of the 'ancient' actin turnover machineries is limited. We show that, despite > 1 billion years of evolution, pathogenic Leishmania major parasite and mammalian actins share the same overall fold and co-polymerize with each other. Interestingly, Leishmania harbors a simple actin-regulatory machinery that lacks cofilin 'cofactors', which accelerate filament disassembly in higher eukaryotes. By applying single-filament biochemistry we discovered that, compared to mammalian proteins, Leishmania actin filaments depolymerize more rapidly from both ends, and are severed > 100-fold more efficiently by cofilin. Our high-resolution cryo-EM structures of Leishmania ADP-, ADP-Pi- and cofilin-actin filaments identify specific features at actin subunit interfaces and cofilin-actin interactions that explain the unusually rapid dynamics of parasite actin filaments. Our findings reveal how divergent parasites achieve rapid actin dynamics using a remarkably simple set of actin-binding proteins, and elucidate evolution of the actin cytoskeleton.

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.

Cellular organization in lab-evolved and extant multicellular species obeys a maximum entropy law.

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri. We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing.

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.

The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

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.

Dynamics of Tpm1.8 domains on actin filaments with single-molecule resolution.

Tropomyosins regulate the dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of the fluorescently labeled tropomyosin isoform Tpm1.8 to unlabeled actin filaments in real time. This approach, in conjunction with mathematical modeling, enabled us to quantify the nucleation, assembly, and disassembly kinetics of Tpm1.8 on single filaments and at the single-molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilized by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented toward the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Actin reduction by MsrB2 is a key component of the cytokinetic abscission checkpoint and prevents tetraploidy.

Abscission is the terminal step of cytokinesis leading to the physical separation of the daughter cells. In response to the abnormal presence of lagging chromatin between dividing cells, an evolutionarily conserved abscission/NoCut checkpoint delays abscission and prevents formation of binucleated cells by stabilizing the cytokinetic intercellular bridge (ICB). How this bridge is stably maintained for hours while the checkpoint is activated is poorly understood and has been proposed to rely on F-actin in the bridge region. Here, we show that actin polymerization is indeed essential for stabilizing the ICB when lagging chromatin is present, but not in normal dividing cells. Mechanistically, we found that a cytosolic pool of human methionine sulfoxide reductase B2 (MsrB2) is strongly recruited at the midbody in response to the presence of lagging chromatin and functions within the ICB to promote actin polymerization there. Consistently, in MsrB2-depleted cells, F-actin levels are decreased in ICBs, and dividing cells with lagging chromatin become binucleated as a consequence of unstable bridges. We further demonstrate that MsrB2 selectively reduces oxidized actin monomers and thereby counteracts MICAL1, an enzyme known to depolymerize actin filaments by direct oxidation. Finally, MsrB2 colocalizes and genetically interacts with the checkpoint components Aurora B and ANCHR, and the abscission delay upon checkpoint activation by nuclear pore defects also depends on MsrB2. Altogether, this work reveals that actin reduction by MsrB2 is a key component of the abscission checkpoint that favors F-actin polymerization and limits tetraploidy, a starting point for tumorigenesis.

The advantages of microfluidics to study actin biochemistry and biomechanics.

The regulated assembly of actin filaments is essential in nearly all cell types. Studying actin assembly dynamics can pose many technical challenges. A number of these challenges can be overcome by using microfluidics to observe and manipulate single actin filaments under an optical microscope. In particular, microfluidics can be tremendously useful for applying different mechanical stresses to actin filaments and determining how the physical context of the filaments affects their regulation by biochemical factors. In this review, we summarize the main features of microfluidics for the study of actin assembly dynamics, and we highlight some recent developments that have emerged from the combination of microfluidics and other techniques. We use two case studies to illustrate our points: the rapid assembly of actin filaments by formins and the disassembly of filaments by actin depolymerizing factor (ADF)/cofilin. Both of these protein families play important roles in cells. They regulate actin assembly through complex molecular mechanisms that are sensitive to the filaments' mechanical context, with multiple activities that need to be quantified separately. Microfluidics-based experiments have been extremely useful for gaining insight into the regulatory actions of these two protein families.

Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin.

The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.

Torsional stress generated by ADF/cofilin on cross-linked actin filaments boosts their severing.

Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin's ability to sever highly connected filament networks in cells.

Quantitative Variations with pH of Actin Depolymerizing Factor/Cofilin's Multiple Actions on Actin Filaments.

Actin depolymerizing factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, at four different pH values ranging from 6.6 to 7.8, using single-filament microfluidics techniques. We show that decreasing the pH decreases the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing rate per domain boundary, however, remains unchanged at different pH values. The ADF/cofilin-decorated filaments ("cofilactin" filaments) depolymerize from both ends. We show here that, at physiological pH (7.0-7.4), the pointed end depolymerization of cofilactin filaments is barely faster than that of bare filaments. In contrast, cofilactin barbed ends undergo an "unstoppable" depolymerization (depolymerizing for minutes despite the presence of free actin monomers and capping protein in solution), throughout our pH range. We thus show that, at physiological pH, the main contribution of ADF/cofilin to filament depolymerization is at the barbed end.

Modulation of formin processivity by profilin and mechanical tension.

Formins are major regulators of actin networks. They enhance actin filament dynamics by remaining processively bound to filament barbed ends. How biochemical and mechanical factors affect formin processivity are open questions. Monitoring individual actin filaments in a microfluidic flow, we report that formins mDia1 and mDia2 dissociate faster under higher ionic strength and when actin concentration is increased. Profilin, known to increase the elongation rate of formin-associated filaments, surprisingly decreases the formin dissociation rate, by bringing formin FH1 domains in transient contact with the barbed end. In contrast, piconewton tensile forces applied to actin filaments accelerate formin dissociation by orders of magnitude, largely overcoming profilin-mediated stabilization. We developed a model of formin conformations showing that our data indicates the existence of two different dissociation pathways, with force favoring one over the other. How cells limit formin dissociation under tension is now a key question for future studies.

ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends.

Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells.

Oxidation of F-actin controls the terminal steps of cytokinesis.

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division.

Ferromagnetic and antiferromagnetic order in bacterial vortex lattices.

Despite their inherent non-equilibrium nature1, living systems can self-organize in highly ordered collective states2,3 that share striking similarities with the thermodynamic equilibrium phases4,5 of conventional condensed matter and fluid systems. Examples range from the liquid-crystal-like arrangements of bacterial colonies6,7, microbial suspensions8,9 and tissues10 to the coherent macro-scale dynamics in schools of fish11 and flocks of birds12. Yet, the generic mathematical principles that govern the emergence of structure in such artificial13 and biological6-9,14 systems are elusive. It is not clear when, or even whether, well-established theoretical concepts describing universal thermostatistics of equilibrium systems can capture and classify ordered states of living matter. Here, we connect these two previously disparate regimes: Through microfluidic experiments and mathematical modelling, we demonstrate that lattices of hydrodynamically coupled bacterial vortices can spontaneously organize into distinct phases of ferro- and antiferromagnetic order. The preferred phase can be controlled by tuning the vortex coupling through changes of the inter-cavity gap widths. The emergence of opposing order regimes is tightly linked to the existence of geometry-induced edge currents15,16, reminiscent of those in quantum systems17-19. Our experimental observations can be rationalized in terms of a generic lattice field theory, suggesting that bacterial spin networks belong to the same universality class as a wide range of equilibrium systems.

Fluid flows created by swimming bacteria drive self-organization in confined suspensions.

Concentrated suspensions of swimming microorganisms and other forms of active matter are known to display complex, self-organized spatiotemporal patterns on scales that are large compared with those of the individual motile units. Despite intensive experimental and theoretical study, it has remained unclear the extent to which the hydrodynamic flows generated by swimming cells, rather than purely steric interactions between them, drive the self-organization. Here we use the recent discovery of a spiral-vortex state in confined suspensions of Bacillus subtilis to study this issue in detail. Those experiments showed that if the radius of confinement in a thin cylindrical chamber is below a critical value, the suspension will spontaneously form a steady single-vortex state encircled by a counter-rotating cell boundary layer, with spiral cell orientation within the vortex. Left unclear, however, was the flagellar orientation, and hence the cell swimming direction, within the spiral vortex. Here, using a fast simulation method that captures oriented cell-cell and cell-fluid interactions in a minimal model of discrete particle systems, we predict the striking, counterintuitive result that in the presence of collectively generated fluid motion, the cells within the spiral vortex actually swim upstream against those flows. This prediction is then confirmed by the experiments reported here, which include measurements of flagella bundle orientation and cell tracking in the self-organized state. These results highlight the complex interplay between cell orientation and hydrodynamic flows in concentrated suspensions of microorganisms.