Focal adhesions contain three specialized actin nanoscale layers.

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.

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.

Recessive TMOD1 mutation causes childhood cardiomyopathy.

Familial cardiomyopathy in pediatric stages is a poorly understood presentation of heart disease in children that is attributed to pathogenic mutations. Through exome sequencing, we report a homozygous variant in tropomodulin 1 (TMOD1; c.565C>T, p.R189W) in three individuals from two unrelated families with childhood-onset dilated and restrictive cardiomyopathy. To decipher the mechanism of pathogenicity of the R189W mutation in TMOD1, we utilized a wide array of methods, including protein analyses, biochemistry and cultured cardiomyocytes. Structural modeling revealed potential defects in the local folding of TMOD1R189W and its affinity for actin. Cardiomyocytes expressing GFP-TMOD1R189W demonstrated longer thin filaments than GFP-TMOD1wt-expressing cells, resulting in compromised filament length regulation. Furthermore, TMOD1R189W showed weakened activity in capping actin filament pointed ends, providing direct evidence for the variant's effect on actin filament length regulation. Our data indicate that the p.R189W variant in TMOD1 has altered biochemical properties and reveals a unique mechanism for childhood-onset cardiomyopathy.

Single particle tracking reveals SARS-CoV-2 regulating and utilizing dynamic filopodia for viral invasion.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the subcellular structural remodeling to invade multiple organs and cell types. Here, we unveil how SARS-CoV-2 boosts and utilizes filopodia to enter the target cells by real-time imaging. Using SARS-CoV-2 single virus-like particle (VLP) tracking in live cells and sparse deconvolution algorithm, we uncover that VLPs utilize filopodia to reach the entry site in two patterns, "surfing" and "grabbing", which avoid the virus from randomly searching on the plasma membrane. Moreover, combining mechanical simulation, we elucidate that the formation of virus-induced filopodia and the retraction speed of filopodia depend on cytoskeleton dynamics and friction resistance at the substrate surface caused by loading-virus gravity, respectively. Further, we discover that the entry process of SARS-CoV-2 via filopodia depends on Cdc42 activity and actin-associated proteins fascin, formin, and Arp2/3. Together, our results highlight that the spatial-temporal regulation of actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry and potentiates it as an antiviral target.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Recycling of the actin monomer pool limits the lifetime of network turnover.

Intracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks.

Structural basis underlying specific biochemical activities of non-muscle tropomyosin isoforms.

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the actin filament major groove, providing an explanation for their incapability to co-polymerize on actin filaments. We also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalyzed severing.

Caldesmon controls stress fiber force-balance through dynamic cross-linking of myosin II and actin-tropomyosin filaments.

Contractile actomyosin bundles are key force-producing and mechanosensing elements in muscle and non-muscle tissues. Whereas the organization of muscle myofibrils and mechanism regulating their contractility are relatively well-established, the principles by which myosin-II activity and force-balance are regulated in non-muscle cells have remained elusive. We show that Caldesmon, an important component of smooth muscle and non-muscle cell actomyosin bundles, is an elongated protein that functions as a dynamic cross-linker between myosin-II and tropomyosin-actin filaments. Depletion of Caldesmon results in aberrant lateral movement of myosin-II filaments along actin bundles, leading to irregular myosin distribution within stress fibers. This manifests as defects in stress fiber network organization and contractility, and accompanied problems in cell morphogenesis, migration, invasion, and mechanosensing. These results identify Caldesmon as critical factor that ensures regular myosin-II spacing within non-muscle cell actomyosin bundles, and reveal how stress fiber networks are controlled through dynamic cross-linking of tropomyosin-actin and myosin filaments.

Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions.

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

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.

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.

Mechanism of Borrelia immune evasion by FhbA-related proteins.

Immune evasion facilitates survival of Borrelia, leading to infections like relapsing fever and Lyme disease. Important mechanism for complement evasion is acquisition of the main host complement inhibitor, factor H (FH). By determining the 2.2 Å crystal structure of Factor H binding protein A (FhbA) from Borrelia hermsii in complex with FH domains 19-20, combined with extensive mutagenesis, we identified the structural mechanism by which B. hermsii utilizes FhbA in immune evasion. Moreover, structure-guided sequence database analysis identified a new family of FhbA-related immune evasion molecules from Lyme disease and relapsing fever Borrelia. Conserved FH-binding mechanism within the FhbA-family was verified by analysis of a novel FH-binding protein from B. duttonii. By sequence analysis, we were able to group FH-binding proteins of Borrelia into four distinct phyletic types and identified novel putative FH-binding proteins. The conserved FH-binding mechanism of the FhbA-related proteins could aid in developing new approaches to inhibit virulence and complement resistance in Borrelia.

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair.

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. Although biochemical mechanisms that regulate the activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM-II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein level of UNC-45A was decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29cf8 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at apical junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

An ARHGAP25 variant links aberrant Rac1 function to early-onset skeletal fragility.

Ras homologous guanosine triphosphatases (RhoGTPases) control several cellular functions, including cytoskeletal actin remodeling and cell migration. Their activities are downregulated by GTPase-activating proteins (GAPs). Although RhoGTPases are implicated in bone remodeling and osteoclast and osteoblast function, their significance in human bone health and disease remains elusive. Here, we report defective RhoGTPase regulation as a cause of severe, early-onset, autosomal-dominant skeletal fragility in a three-generation Finnish family. Affected individuals (n = 13) presented with multiple low-energy peripheral and vertebral fractures despite normal bone mineral density (BMD). Bone histomorphometry suggested reduced bone volume, low surface area covered by osteoblasts and osteoclasts, and low bone turnover. Exome sequencing identified a novel heterozygous missense variant c.652G>A (p.G218R) in ARHGAP25, encoding a GAP for Rho-family GTPase Rac1. Variants in the ARHGAP25 5' untranslated region (UTR) also associated with BMD and fracture risk in the general population, across multiple genomewide association study (GWAS) meta-analyses (lead variant rs10048745). ARHGAP25 messenger RNA (mRNA) was expressed in macrophage colony-stimulating factor (M-CSF)-stimulated human monocytes and mouse osteoblasts, indicating a possible role for ARHGAP25 in osteoclast and osteoblast differentiation and activity. Studies on subject-derived osteoclasts from peripheral blood mononuclear cells did not reveal robust defects in mature osteoclast formation or resorptive activity. However, analysis of osteosarcoma cells overexpressing the ARHGAP25 G218R-mutant, combined with structural modeling, confirmed that the mutant protein had decreased GAP-activity against Rac1, resulting in elevated Rac1 activity, increased cell spreading, and membrane ruffling. Our findings indicate that mutated ARHGAP25 causes aberrant Rac1 function and consequently abnormal bone metabolism, highlighting the importance of RhoGAP signaling in bone metabolism in familial forms of skeletal fragility and in the general population, and expanding our understanding of the molecular pathways underlying skeletal fragility. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.