Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

Optical Control of G-Actin with a Photoswitchable Latrunculin.

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Myosin-5 varies its step length to carry cargo straight along the irregular F-actin track.

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.

Memory induced-mechanism of noise attenuator of myosin V molecular motors.

Depending on the chemical energy from ATP hydrolysis, myosin V can drive the multistep and continuous coupled cycling process to transport cellular cargo to targeted regions. However, it is still obscure how the molecular memory induced by the multistep coupled transported process could regulate the dynamic behavior of the motor state of myosin V. Here, we propose a novel non-Markovian polymorphic mechanochemical model to investigate the effect of the molecular memory on the mechanic of noise attenuation of myosin V system. We first define an effective transition rate for a multistep coupled reaction process which is the function of memory and system states to transform equivalently the non-Markovian process into the classical Markov process. By noise decomposition technology, it is observed that both the intrinsic and extrinsic noises of the ADP-myosin V bound state (AM ⋅ ADP) exhibit a monotonically decreasing trend with lengthening the molecular memory. Molecular memory as a regulation factor can amplify the contribution of intrinsic noise to the overall noise while reducing the influence of extrinsic noise on the AM ⋅ ADP. Moreover, the modulation of molecular memory could induce stochastic focusing. These results indicate that the role of molecular memory in the myosin V state transition can not only offer a handle to maintain the robustness of the motion system but also serve as a paradigm for studying more complex molecular motors.

Dynamically Mapping the Topography and Stiffness of the Leading Edge of Migrating Cells Using AFM in Fast-QI Mode.

Cell migration profoundly influences cellular function, often resulting in adverse effects in various pathologies including cancer metastasis. Directly assessing and quantifying the nanoscale dynamics of living cell structure and mechanics has remained a challenge. At the forefront of cell movement, the flat actin modules─the lamellipodium and the lamellum─interact to propel cell migration. The lamellipodium extends from the lamellum and undergoes rapid changes within seconds, making measurement of its stiffness a persistent hurdle. In this study, we introduce the fast-quantitative imaging (fast-QI) mode, demonstrating its capability to simultaneously map both the lamellipodium and the lamellum with enhanced spatiotemporal resolution compared with the classic quantitative imaging (QI) mode. Specifically, our findings reveal nanoscale stiffness gradients in the lamellipodium at the leading edge, where it appears to be slightly thinner and significantly softer than the lamellum. Additionally, we illustrate the fast-QI mode's accuracy in generating maps of height and effective stiffness through a streamlined and efficient processing of force-distance curves. These results underscore the potential of the fast-QI mode for investigating the role of motile cell structures in mechanosensing.

Sub-membrane actin rings compartmentalize the plasma membrane.

The compartmentalization of the plasma membrane (PM) is a fundamental feature of cells. The diffusivity of membrane proteins is significantly lower in biological than in artificial membranes. This is likely due to actin filaments, but assays to prove a direct dependence remain elusive. We recently showed that periodic actin rings in the neuronal axon initial segment (AIS) confine membrane protein motion between them. Still, the local enrichment of ion channels offers an alternative explanation. Here we show, using computational modeling, that in contrast to actin rings, ion channels in the AIS cannot mediate confinement. Furthermore, we show, employing a combinatorial approach of single particle tracking and super-resolution microscopy, that actin rings are close to the PM and that they confine membrane proteins in several neuronal cell types. Finally, we show that actin disruption leads to loss of compartmentalization. Taken together, we here develop a system for the investigation of membrane compartmentalization and show that actin rings compartmentalize the PM.

Fascin - F-actin interaction studied by molecular dynamics simulation and protein network analysis.

Actin bundles are an important component of cellular cytoskeleton and participate in the movement of cells. The formation of actin bundles requires the participation of many actin binding proteins (ABPs). Fascin is a member of ABPs, which plays a key role in bundling filamentous actin (F-actin) to bundles. However, the detailed interactions between fascin and F-actin are unclear. In this study, we construct an atomic-level structure of fascin - F-actin complex based on a rather poor cryo-EM data with resolution of 20 nm. We first optimized the geometries of the complex by molecular dynamics (MD) simulation and analyzed the binding site and pose of fascin which bundles two F-actin chains. Next, binding free energy of fascin was calculated by MM/GBSA method. Finally, protein structure network analysis (PSNs) was performed to analyze the key residues for fascin binding. Our results show that residues of K22, E27, E29, K41, K43, R110, R149, K358, R408 and K471 on fascin are important for its bundling, which are in good agreement with the experimental data. On the other hand, the consistent results indicate that the atomic-level model of fascin - F-actin complex is reliable. In short, this model can be used to understand the detailed interactions between fascin and F-actin, and to develop novel potential drugs targeting fascin.Communicated by Ramaswamy H. Sarma.

Interactions between paramyosin and actin greatly improve their thermostability and gel properties.

BACKGROUND: Myofibrillar proteins, the main contributors to the quality of meat products, are the main structural protein component of muscle and have functional properties such as the formation of a 3D protein gel network and water binding. The susceptibility of meat-derived proteins to heat-induced aggregation is the functional constraint that hinders their applications in industry, and so establishing an effective but simple method to improve their thermostability of the proteins is of great importance. RESULTS: In the present study, we describe an easy approach to perform high colloidal thermostability of both paramyosin and actin by mixing them at low ionic strength. The improvement in thermal stability was found to be derived from intermolecular interactions between these two different proteins through non-covalent binding with each other. Consequently, such interactions protected each of them from thermal-induced degradation compared to individual components. Notably, this binary native protein mixture rather than single paramyosin or actin component has the ability to form protein hydrogels with a shear-thinning and reversible sol-gel transformation behavior, which is markedly different from most of reported heat-induced, denatured protein hydrogels. CONCLUSION: The present study not only presents a facile and effective strategy for improvement of the thermal stability and gel properties of a binary paramyosin and actin mixture, but also enhances our understanding of how mutual interactions of protein components affect their physicochemical and functional properties. © 2023 Society of Chemical Industry.

Drebrin Protects Assembled Actin from INF2-FFC-mediated Severing and Stabilizes Cell Protrusions.

Highly specialized cells, such as neurons and podocytes, have arborized morphologies that serve their specific functions. Actin cytoskeleton and its associated proteins are responsible for the distinctive shapes of cells. The mechanism of their cytoskeleton regulation - contributing to cell shape maintenance - is yet to be fully clarified. Inverted formin 2 (INF2), one of the modulators of the cytoskeleton, is an atypical formin that can both polymerize and depolymerize actin filaments depending on its molar ratio to actin. Prior work has established that INF2 binds to the sides of actin filaments and severs them. Drebrin is another actin-binding protein that also binds filaments laterally and stabilizes them, but the interplay between drebrin and INF2 on actin filament stabilization is not well understood. Here, we have used biochemical assays, electron microscopy, and total internal reflection fluorescence microscopy imaging to show that drebrin protects actin filaments from severing by INF2 without inhibiting its polymerization activity. Notably, truncated drebrin - DrbA1-300 - is sufficient for this protection, though not as effective as the full-length protein. INF2 and drebrin are abundantly expressed in highly specialized cells and are crucial for the temporal regulation of their actin cytoskeleton, consistent with their involvement in peripheral neuropathy.

Transverse Fluctuations Control the Assembly of Semiflexible Filaments.

The kinetics of the assembly of semiflexible filaments through end-to-end annealing is key to the structure of the cytoskeleton, but is not understood. We analyze this problem through scaling theory and simulations, and uncover a regime where filaments' ends find each other through bending fluctuations without the need for the whole filament to diffuse. This results in a very substantial speedup of assembly in physiological regimes, and could help with understanding the dynamics of actin and intermediate filaments in biological processes such as wound healing and cell division.

Cyclase-associated protein interacts with actin filament barbed ends to promote depolymerization and formin displacement.

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.

The Role of Trp79 in ß-Actin on Histidine Methyltransferase SETD3 Catalysis.

Nτ -methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in ß-actin on productive human SETD3 catalysis. Substitution of Trp79 in ß-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.

Divalent ions as mediators of carbonylation in cardiac myosin binding protein C.

The dosing and efficacy of chemotherapeutic drugs can be limited by toxicity caused by off-pathway reactions. One hypothesis for how such toxicity arises is via metal-catalyzed oxidative damage of cardiac myosin binding protein C (cMyBP-C) found in cardiac tissue. Previous research indicates that metal ion mediated reactive oxygen species induce high levels of protein carbonylation, changing the structure and function of this protein. In this work, we use long timescale all-atom molecular dynamics simulations to investigate the ion environment surrounding the C0 and C1 subunits of cMyBP-C responsible for actin binding. We show that divalent cations are co-localized with protein carbonylation-prone amino acid residues and that carbonylation of these residues can lead to site-specific interruption to the actin-cMyBP-C binding.

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor.

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and ß subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles.

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.

Structural and Functional Properties of Kappa Tropomyosin.

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Structures of the free and capped ends of the actin filament.

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Functional comparison of full-length palladin to isolated actin binding domain.

Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline-rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F-actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full-length palladin in actin binding stoichiometry, polymerization, and interactions with G-actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.