LUZP1 regulates the maturation of contractile actomyosin bundles.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

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.

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.

Scarcity and panic buying: the effect of regulation by subsidizing the supply and customer purchases during a crisis.

During the first wave of the COVID-19 pandemic, in France, people cleared the shelves of butter; in Italy, it was pasta; in Great Britain, it was chicken. While there may be cultural disagreement on what is essential, clearly, in times of crisis, consumers stockpile the 'essentials'. We address the problem of "panic buying", which is characterized by increasing demand in the face of diminishing inventory. In such cases, prices may hike and firms (retailers) selling the high-demand product are quantity takers, in terms of supply, and price setters. We consider a manufacturer who sells a scarce product to a single retailer. The retailer seeks to maximize her profit, while in contrast, the manufacturer pursues a social objective of regulating and lowering the amount that the end customer (consumer) pays (including the cost of traveling to obtain the scarce product). By analyzing the competition between the two parties, retailer and manufacturer, we find that even when the regulator (manufacturer) makes a significant social commitment, neither subsidizing the retailer nor subsidizing the consumers necessarily curbs price hikes. Furthermore, there is a threshold ratio (i.e., proportion of the end price subsidized by the regulator) that determines the minimal budget that the regulator would need to allocate in order for subsidization to make a difference to consumers.

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.

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.

A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics.

Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.

Tropomodulins Control the Balance between Protrusive and Contractile Structures by Stabilizing Actin-Tropomyosin Filaments.

Eukaryotic cells have diverse protrusive and contractile actin filament structures, which compete with one another for a limited pool of actin monomers. Numerous actin-binding proteins regulate the dynamics of actin structures, including tropomodulins (Tmods), which cap the pointed end of actin filaments. In striated muscles, Tmods prevent actin filaments from overgrowing, whereas in non-muscle cells, their function has remained elusive. Here, we identify two Tmod isoforms, Tmod1 and Tmod3, as key components of contractile stress fibers in non-muscle cells. Individually, Tmod1 and Tmod3 can compensate for one another, but their simultaneous depletion results in disassembly of actin-tropomyosin filaments, loss of force-generating stress fibers, and severe defects in cell morphology. Knockout-rescue experiments reveal that Tmod's interaction with tropomyosin is essential for its role in the stabilization of actin-tropomyosin filaments in cells. Thus, in contrast to their role in muscle myofibrils, in non-muscle cells, Tmods bind actin-tropomyosin filaments to protect them from depolymerizing, not elongating. Furthermore, loss of Tmods shifts the balance from linear actin-tropomyosin filaments to Arp2/3 complex-nucleated branched networks, and this phenotype can be partially rescued by inhibiting the Arp2/3 complex. Collectively, the data reveal that Tmods are essential for the maintenance of contractile actomyosin bundles and that Tmod-dependent capping of actin-tropomyosin filaments is critical for the regulation of actin homeostasis in non-muscle cells.

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.

Calponin-3 is critical for coordinated contractility of actin stress fibers.

Contractile actomyosin bundles, stress fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), stress fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with stress fibers. However, whether Cnn3 promotes stress fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral stress fibers and transverse arcs, as well as to non-contractile dorsal stress fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of stress fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for stress fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of stress fibers that often led to breakage of individual actomyosin bundles within the stress fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the stress fiber network.

Part-time practice in healthcare: Impact on operational versus medical performance.

We explore how reliance on part-time medical staff affects operational and medical outcome performance in two general surgery departments Whereas prior research has indicated that operational performance is positively associated with medical performance, we find that heavier reliance on part-time practice may deteriorate operational performance but not necessarily medical-outcome performance. For so-called "complex" patients, reliance on part-time practice may even override the effect of patients' characteristics on medical-outcome performance. This result calls into question common perceptions in behavior marketing literature regarding part-time employees' working patterns and efficiency, and thereby provides a new perspective regarding current labor-market trends.

Structural basis of actin monomer re-charging by cyclase-associated protein.

Actin polymerization powers key cellular processes, including motility, morphogenesis, and endocytosis. The actin turnover cycle depends critically on "re-charging" of ADP-actin monomers with ATP, but whether this reaction requires dedicated proteins in cells, and the underlying mechanism, have remained elusive. Here we report that nucleotide exchange catalyzed by the ubiquitous cytoskeletal regulator cyclase-associated protein (CAP) is critical for actin-based processes in vivo. We determine the structure of the CAP-actin complex, which reveals that nucleotide exchange occurs in a compact, sandwich-like complex formed between the dimeric actin-binding domain of CAP and two ADP-actin monomers. In the crystal structure, the C-terminal tail of CAP associates with the nucleotide-sensing region of actin, and this interaction is required for rapid re-charging of actin by both yeast and mammalian CAPs. These data uncover the conserved structural basis and biological role of protein-catalyzed re-charging of actin monomers.

Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase "RET", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 µM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.

Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.

The structure and catalytic cycle of a sodium-pumping pyrophosphatase.

Membrane-integral pyrophosphatases (M-PPases) are crucial for the survival of plants, bacteria, and protozoan parasites. They couple pyrophosphate hydrolysis or synthesis to Na(+) or H(+) pumping. The 2.6-angstrom structure of Thermotoga maritima M-PPase in the resting state reveals a previously unknown solution for ion pumping. The hydrolytic center, 20 angstroms above the membrane, is coupled to the gate formed by the conserved Asp(243), Glu(246), and Lys(707) by an unusual "coupling funnel" of six α helices. Comparison with our 4.0-angstrom resolution structure of the product complex suggests that helix 12 slides down upon substrate binding to open the gate by a simple binding-change mechanism. Below the gate, four helices form the exit channel. Superimposing helices 3 to 6, 9 to 12, and 13 to 16 suggests that M-PPases arose through gene triplication.