Structural basis of human Nav1.5 gating mechanisms.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

Competition between myosin II and ßH-spectrin regulates cytoskeletal tension.

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two ß-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila ß-heavy spectrin (ßH-spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that ßH-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that ßH-spectrin localizes and functions independently of α-spectrin. Instead, ßH-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which ßH-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of ßH-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how ßH-spectrin participates in ratcheting mechanisms associated with cell shape change.

Structural insights into actin isoforms.

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle differences in their design principles determine the interaction with myosin motors and actin-binding proteins. Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the Mg2+·ADP state with their native post-translational modifications. The structures revealed isoform-specific conformations of the N-terminus that shift closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Collectively, the structures of single-isotype, post-translationally modified bare skeletal muscle α-actin, cardiac muscle α-actin, ß-actin, and γ-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

The protein actin is important for many fundamental processes in biology, from contracting muscle to dividing a cell in two. As actin is involved in such a variety of roles, human cells have slightly different versions of the protein, known as isoforms. For example, alpha-actin is vital for contracting muscle, while beta- and gamma-actin drive cellular processes in non-muscle cells. In order to carry out its various functions, actin interacts with many other proteins inside the cell, such as myosin motors which power muscle contraction. These interactions rely on the precise chain of building blocks, known as amino acids, that make up the actin isoforms; even subtle alterations in this sequence can influence the behavior of the protein. However, it is not clear how differences in the amino acid sequence of the actin isoforms impact actin's interactions with other proteins. Arora et al. addressed this by studying the structure of four human actin isoforms using a technique called cryo-electron microscopy, where the proteins are flash-frozen and bombarded with electrons. These experiments showed where differences between the amino acid chains of each isoform were located in the protein. Arora et al. then compared their structures with previous work showing the structure of actin bound to myosin. This revealed that the tail-end of the protein (known as the N-terminus) differed in shape between the four isoforms, and this variation may influence how actin binds to others proteins in the cell. These results are an important foundation for further work on actin and how it interacts with other proteins. The structures could help researchers design new tools that can be used to target specific isoforms of actin in different types of laboratory experiments.

Photorhabdus luminescens TccC3 Toxin Targets the Dynamic Population of F-Actin and Impairs Cell Cortex Integrity.

Due to its essential role in cellular processes, actin is a common target for bacterial toxins. One such toxin, TccC3, is an effector domain of the ABC-toxin produced by entomopathogenic bacteria of Photorhabdus spp. Unlike other actin-targeting toxins, TccC3 uniquely ADP-ribosylates actin at Thr-148, resulting in the formation of actin aggregates and inhibition of phagocytosis. It has been shown that the fully modified F-actin is resistant to depolymerization by cofilin and gelsolin, but their effects on partially modified actin were not explored. We found that only F-actin unprotected by tropomyosin is the physiological TccC3 substrate. Yet, ADP-ribosylated G-actin can be produced upon cofilin-accelerated F-actin depolymerization, which was only mildly inhibited in partially modified actin. The affinity of TccC3-ADP-ribosylated G-actin for profilin and thymosin-ß4 was weakened moderately but sufficiently to potentiate spontaneous polymerization in their presence. Interestingly, the Arp2/3-mediated nucleation was also potentiated by T148-ADP-ribosylation. Notably, even partially modified actin showed reduced bundling by plastins and α-actinin. In agreement with the role of these and other tandem calponin-homology domain actin organizers in the assembly of the cortical actin network, TccC3 induced intense membrane blebbing in cultured cells. Overall, our data suggest that TccC3 imposes a complex action on the cytoskeleton by affecting F-actin nucleation, recycling, and interaction with actin-binding proteins involved in the integration of actin filaments with each other and cellular elements.

MARK2 regulates directed cell migration through modulation of myosin II contractility and focal adhesion organization.

Cancer cell migration during metastasis is mediated by a highly polarized cytoskeleton. MARK2 and its invertebrate homolog Par1B are kinases that regulate the microtubule cytoskeleton to mediate polarization of neurons in mammals and embryos in invertebrates. However, the role of MARK2 in cancer cell migration is unclear. Using osteosarcoma cells, we found that in addition to its known localizations on microtubules and the plasma membrane, MARK2 also associates with the actomyosin cytoskeleton and focal adhesions. Cells depleted of MARK proteins demonstrated that MARK2 promotes phosphorylation of both myosin II and the myosin phosphatase targeting subunit MYPT1 to synergistically drive myosin II contractility and stress fiber formation in cells. Studies with isolated proteins showed that MARK2 directly phosphorylates myosin II regulatory light chain, while its effects on MYPT1 phosphorylation are indirect. Using a mutant lacking the membrane-binding domain, we found that membrane association is required for focal adhesion targeting of MARK2, where it specifically enhances cell protrusion by promoting FAK phosphorylation and formation of focal adhesions oriented in the direction of migration to mediate directionally persistent cell motility. Together, our results define MARK2 as a master regulator of the actomyosin and microtubule cytoskeletal systems and focal adhesions to mediate directional cancer cell migration.

Cryo-EM structure of the autoinhibited state of myosin-2.

We solved the near-atomic resolution structure of smooth muscle myosin-2 in the autoinhibited state (10S) using single-particle cryo­electron microscopy. The 3.4-Å structure reveals the precise molecular architecture of 10S and the structural basis for myosin-2 regulation. We reveal the position of the phosphorylation sites that control myosin autoinhibition and activation by phosphorylation of the regulatory light chain. Further, we present a previously unidentified conformational state in myosin-2 that traps ADP and Pi produced by the hydrolysis of ATP in the active site. This noncanonical state represents a branch of the myosin enzyme cycle and explains the autoinhibition of the enzyme function of 10S along with its reduced affinity for actin. Together, our structure defines the molecular mechanisms that drive 10S formation, stabilization, and relief by phosphorylation of the regulatory light chain.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness.

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (kcat = ∼ 6 s-1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s-1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells.

Active non-muscle myosin II (NMII) enables migratory cell polarization and controls dynamic cellular processes, such as focal adhesion formation and turnover and cell division. Filament assembly and force generation depend on NMII activation through the phosphorylation of Ser19 of the regulatory light chain (RLC). Here, we identify amino acid Tyr (Y) 155 of the RLC as a novel regulatory site that spatially controls NMII function. We show that Y155 is phosphorylated in vitro by the Tyr kinase domain of epidermal growth factor (EGF) receptor. In cells, phosphorylation of Y155, or its phospho-mimetic mutation (Glu), prevents the interaction of RLC with the myosin heavy chain (MHCII) to form functional NMII units. Conversely, Y155 mutation to a structurally similar but non-phosphorylatable amino acid (Phe) restores the more dynamic cellular functions of NMII, such as myosin filament formation and nascent adhesion assembly, but not those requiring stable actomyosin bundles, e.g., focal adhesion elongation or migratory front-back polarization. In live cells, phospho-Y155 RLC is prominently featured in protrusions, where it prevents NMII assembly. Our data indicate that Y155 phosphorylation constitutes a novel regulatory mechanism that contributes to the compartmentalization of NMII assembly and function in live cells.

The BAR domain of the Arf GTPase-activating protein ASAP1 directly binds actin filaments.

The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.

Nonmuscle myosin-2 isoforms.

A hallmark of eukaryotes is the ability to generate microscale movements from the molecular to the tissue-length scales. Members of the myosin and actin protein families form the actomyosin cytoskeleton and are responsible for these movements. The cytoskeleton is a diffuse and dynamic network made up of different actin structures including arcs, bundles and single filaments, which are often associated with actin regulatory proteins. As such, it spans the entire cell and provides it with structural support.

Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level.

Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors. Despite its fundamental role in recycling endosome trafficking and in collective actin network dynamics, the molecular mechanisms underlying its motility are inherently unknown. Here we combine single-molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B. We show that a single myosin-5B moves processively in 36-nm steps, stalls at ~2 pN resistive forces, and reverses its directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity differs from that of myosin-5A, while it is strikingly similar to kinesin-1. In particular, myosin-5B run length is markedly and asymmetrically sensitive to force, a property that might be central to motor ensemble coordination. Furthermore, we show that Ca2+ does not affect the enzymatic activity of the motor unit, but abolishes myosin-5B processivity through calmodulin dissociation, providing important insights into the regulation of postsynaptic cargoes trafficking in neuronal cells.

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

Mechanistic insights into the active site and allosteric communication pathways in human nonmuscle myosin-2C.

Despite a generic, highly conserved motor domain, ATP turnover kinetics and their activation by F-actin vary greatly between myosin-2 isoforms. Here, we present a 2.25 Špre-powerstroke state (ADP⋅VO4) crystal structure of the human nonmuscle myosin-2C motor domain, one of the slowest myosins characterized. In combination with integrated mutagenesis, ensemble-solution kinetics, and molecular dynamics simulation approaches, the structure reveals an allosteric communication pathway that connects the distal end of the motor domain with the active site. Disruption of this pathway by mutation of hub residue R788, which forms the center of a cluster of interactions connecting the converter, the SH1-SH2 helix, the relay helix, and the lever, abolishes nonmuscle myosin-2 specific kinetic signatures. Our results provide insights into structural changes in the myosin motor domain that are triggered upon F-actin binding and contribute critically to the mechanochemical behavior of stress fibers, actin arcs, and cortical actin-based structures.

Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease.

Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5-dependent transport processes in cells.

Re-evaluating the roles of myosin 18Aα and F-actin in determining Golgi morphology.

The peri-centrosomal localization and morphology of the Golgi apparatus depends largely on the microtubule cytoskeleton and the microtubule motor protein dynein. Recent studies proposed that myosin 18Aα (M18Aα) also contributes to Golgi morphology by binding the Golgi protein GOLPH3 and walking along adjacent actin filaments to stretch the Golgi into its classic ribbon structure. Biochemical analyses have shown, however, that M18A is not an actin-activated ATPase and lacks motor activity. Our goal, therefore, was to define the precise molecular mechanism by which M18Aα determines Golgi morphology. We show that purified M18Aα remains inactive in the presence of GOLPH3, arguing against the Golgi-specific activation of the myosin. Using M18A-specific antibodies and expression of GFP-tagged M18Aα, we find no evidence that it localizes to the Golgi. Moreover, several cell lines with reduced or eliminated M18Aα expression exhibited normal Golgi morphology. Interestingly, actin filament disassembly resulted in a marked reduction in lateral stretching of the Golgi in both control and M18Aα-deficient cells. Importantly, this reduction was accompanied by an expansion of the Golgi in the vertical direction, vertical movement of the centrosome, and increases in the height of both the nucleus and the cell. Collectively, our data indicate that M18Aα does not localize to the Golgi or play a significant role in determining its morphology, and suggest that global F-actin disassembly alters Golgi morphology indirectly by altering cell shape.

Drosophila non-muscle myosin II motor activity determines the rate of tissue folding.

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos.

Self-organization of actin networks by a monomeric myosin.

The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase-activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration.

Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution.

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated during a complex mechanochemical reaction cycle. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins, a high-resolution structure of the F-actin­myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release) states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

Kinetic Adaptations of Myosins for Their Diverse Cellular Functions.

Members of the myosin superfamily are involved in all aspects of eukaryotic life. Their function ranges from the transport of organelles and cargos to the generation of membrane tension, and the contraction of muscle. The diversity of physiological functions is remarkable, given that all enzymatically active myosins follow a conserved mechanoenzymatic cycle in which the hydrolysis of ATP to ADP and inorganic phosphate is coupled to either actin-based transport or tethering of actin to defined cellular compartments. Kinetic capacities and limitations of a myosin are determined by the extent to which actin can accelerate the hydrolysis of ATP and the release of the hydrolysis products and are indispensably linked to its physiological tasks. This review focuses on kinetic competencies that - together with structural adaptations - result in myosins with unique mechanoenzymatic properties targeted to their diverse cellular functions.

The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network.

ASAP1 regulates F-actin-based structures and functions, including focal adhesions (FAs) and circular dorsal ruffles (CDRs), cell spreading and migration. ASAP1 function requires its N-terminal BAR domain. We discovered that nonmuscle myosin 2A (NM2A) directly bound the BAR-PH tandem of ASAP1in vitro ASAP1 and NM2A co-immunoprecipitated and colocalized in cells. Knockdown of ASAP1 reduced colocalization of NM2A and F-actin in cells. Knockdown of ASAP1 or NM2A recapitulated each other's effects on FAs, cell migration, cell spreading, and CDRs. The NM2A-interacting BAR domain contributed to ASAP1 control of cell spreading and CDRs. Exogenous expression of NM2A rescued the effect of ASAP1 knockdown on CDRs but ASAP1 did not rescue NM2A knockdown defect in CDRs. Our results support the hypothesis that ASAP1 is a positive regulator of NM2A. Given other binding partners of ASAP1, ASAP1 may directly link signaling and the mechanical machinery of cell migration.