RESUMO
Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in ß-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human ß, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in ß myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in ß myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not ß, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.
Assuntos
Miosinas , Miosinas Ventriculares , Humanos , Miosinas Ventriculares/genética , Miosinas/metabolismo , Adenosina Trifosfatases/metabolismo , Mutação , Actinas/metabolismo , Músculo Esquelético/metabolismoRESUMO
The Anrep effect is an adaptive response that increases left ventricular contractility following an acute rise in afterload. Although the mechanistic origin remains undefined, recent findings suggest a two-phase activation of resting myosin for contraction, involving strain-sensitive and posttranslational phases. We propose that this mobilization represents a transition among the relaxed states of myosin-specifically, from the super-relaxed (SRX) to the disordered-relaxed (DRX)-with DRX myosin ready to participate in force generation. This hypothesis offers a unified explanation that connects myosin's SRX-DRX equilibrium and the Anrep effect as parts of a singular phenomenon. We underscore the significance of this equilibrium in modulating contractility, primarily studied in the context of hypertrophic cardiomyopathy, the most common inherited cardiomyopathy associated with diastolic dysfunction, hypercontractility, and left ventricular hypertrophy. As we posit that the cellular basis of the Anrep effect relies on a two-phased transition of myosin from the SRX to the contraction-ready DRX configuration, any dysregulation in this equilibrium may result in the pathological manifestation of the Anrep phenomenon. For instance, in hypertrophic cardiomyopathy, hypercontractility is linked to a considerable shift of myosin to the DRX state, implying a persistent activation of the Anrep effect. These valuable insights call for additional research to uncover a clinical Anrep fingerprint in pathological states. Here, we demonstrate through noninvasive echocardiographic pressure-volume measurements that this fingerprint is evident in 12 patients with hypertrophic obstructive cardiomyopathy before septal myocardial ablation. This unique signature is characterized by enhanced contractility, indicated by a leftward shift and steepening of the end-systolic pressure-volume relationship, and a prolonged systolic ejection time adjusted for heart rate, which reverses post-procedure. The clinical application of this concept has potential implications beyond hypertrophic cardiomyopathy, extending to other genetic cardiomyopathies and even noncongenital heart diseases with complex etiologies across a broad spectrum of left ventricular ejection fractions.
Assuntos
Cardiomiopatia Hipertrófica , Miosinas , Humanos , Miosinas/metabolismo , Miocárdio/metabolismo , Cardiomiopatia Hipertrófica/patologia , Volume Sistólico , Função Ventricular Esquerda , Contração Miocárdica/fisiologiaRESUMO
Looking back at two decades of research on SPIRE actin nucleator proteins, the first decade was clearly dominated by the discovery of SPIRE proteins as founding members of the novel WH2-domain-based actin nucleators, which initiate actin filament assembly through multiple WH2 actin-binding domains. Through complex formation with formins and class 5 myosins, SPIRE proteins coordinate actin filament assembly and myosin motor-dependent force generation. The discovery of SPIRE-regulated cytoplasmic actin filament meshworks in oocytes initiated the next phase of SPIRE research, which has found that SPIRE proteins are integrated in a diverse range of cell biological processes. In addition to regulating vesicle-based actin filament meshworks, SPIRE proteins function in the organisation of actin structures driving the inward movement of pronuclei of the mouse zygote. Localisation at cortical ring structures and the results of knockdown experiments indicate that SPIRE proteins function in the formation of meiotic cleavage sites in mammalian oocytes and the externalisation of von Willebrand factor from endothelial cells. Alternative splicing targets mammalian SPIRE1 towards mitochondria, where it has a role in fission. In this Review, we summarise the past two decades of SPIRE research by addressing the biochemical and cell biological functions of SPIRE proteins in mammalian reproduction, skin pigmentation and wound healing, as well as in mitochondrial dynamics and host-pathogen interactions.
Assuntos
Actinas , Proteínas dos Microfilamentos , Animais , Camundongos , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células Endoteliais/metabolismo , Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Mamíferos/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function.
Assuntos
Miosinas , Células Vegetais , Miosinas/metabolismo , Células Vegetais/metabolismo , Bryopsida/metabolismo , Bryopsida/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/metabolismo , Marchantia/metabolismo , Marchantia/crescimento & desenvolvimento , Desenvolvimento Vegetal/fisiologiaRESUMO
Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains.
Assuntos
Arabidopsis , Calmodulina , Calmodulina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)-expressing (c-Met+) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. METHODS: In-depth phenotyping of peripheral blood T cells, including c-Met+ T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. RESULTS: We show that c-Met+ T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met+ T cells are distinct from those of c-Met-negative (c-Met-) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met+ T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met+ T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met+ T cells. CONCLUSIONS: Our study demonstrates that the detection of circulating c-Met+ T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.
Assuntos
Doenças Autoimunes , Cardiomiopatias , Miocardite , Humanos , Camundongos , Animais , Autoimunidade , Células T de Memória , Miocardite/etiologia , Miocárdio , Cardiomiopatias/complicações , Miosinas Cardíacas , Inflamação/complicaçõesRESUMO
The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions. This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.
Assuntos
Citoesqueleto de Actina , Miosinas , Actinas/genética , Animais , Bovinos , Forminas , Miosinas/genéticaRESUMO
In response to Ca2+ signals, the evolutionarily-conserved Ca2+ sensor calmodulin (CaM) regulates protein targets via direct interaction. Plants possess many CaM-like (CML) proteins, but their binding partners and functions are mostly unknown. Here, using Arabidopsis CML13 as 'bait' in a yeast two-hybrid screen, we isolated putative targets from three, unrelated protein families, namely, IQD proteins, calmodulin-binding transcriptional activators (CAMTAs) and myosins, all of which possess tandem isoleucine-glutamine (IQ) structural domains. Using the split-luciferase complementation assay in planta and the yeast 2-hybrid system, CML13 and CML14 showed a preference for interaction with tandem over single IQ domains. Relative to CaM, CML13 and CML14 displayed weaker signals when tested with the non-IQ, CaM-binding domain of glutamate decarboxylase or the single IQ domains of CNGC20 (cyclic-nucleotide gated channel-20) or IQM1 (IQ motif protein1). We examined IQD14 as a representative tandem IQ-protein and found that only CaM, CML13 and CML14 interacted with IQD14 among 12 CaM/CMLs tested. CaM, CML13 and CML14 bound in vitro to IQD14 in the presence or absence of Ca2+ . Binding affinities were in the nM range and were higher when two tandem IQ domains from IQD14 were present. Green fluorescent protein-tagged versions of CaM, CML13 and CML14 localized to both the cytosol and nucleus in plant cells but were partially relocalized to the microtubules when co-expressed with IQD14 tagged with mCherry. These and other data are discussed in the context of possible roles for these CMLs in gene regulation via CAMTAs and cytoskeletal activity via myosins and IQD proteins.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Saccharomyces cerevisiae/metabolismo , Sinalização do Cálcio , Ligação Proteica , Cálcio/metabolismoRESUMO
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.
Assuntos
Actomiosina , Miosinas , Actomiosina/metabolismo , Células Epiteliais/metabolismo , Humanos , Junções Intercelulares/metabolismo , Mucosa Intestinal/metabolismo , Chaperonas Moleculares/metabolismo , Miosinas/metabolismo , Junções Íntimas/metabolismoRESUMO
Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics.
Assuntos
Actinas , Cadeias Pesadas de Miosina , Actinas/metabolismo , Endocitose , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/genéticaRESUMO
Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.
Assuntos
Citoesqueleto de Actina/química , Miosina Tipo II/química , Miosinas/química , Miosinas de Músculo Liso/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Galinhas , Microscopia Eletrônica , Miosina Tipo II/genética , Miosina Tipo II/ultraestrutura , Miosinas/genética , Miosinas/ultraestrutura , Fosforilação/genética , Polimerização , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica/genética , Desdobramento de Proteína , Miosinas de Músculo Liso/genética , Miosinas de Músculo Liso/ultraestruturaRESUMO
The inclusion of lymphocytes in high endothelial venules and their migration to the lymph nodes are critical steps in the immune response. Cell migration is regulated by the actin cytoskeleton and myosins. Myo1e is a long-tailed class I myosin and is highly expressed in B cells, which have not been studied in the context of cell migration. By using intravital microscopy in an in vivo model and performing in vitro experiments, we studied the relevance of Myo1e for the adhesion and inclusion of activated B cells in high endothelial venules. We observed reduced expression of integrins and F-actin in the membrane protrusions of B lymphocytes, which might be explained by deficiencies in vesicular trafficking. Interestingly, the lack of Myo1e reduced the phosphorylation of focal adhesion kinase (FAK; also known as PTK2), AKT (also known as AKT1) and RAC-1, disturbing the FAK-PI3K-RAC-1 signaling pathway. Taken together, our results indicate a critical role of Myo1e in the mechanism of B-cell adhesion and migration.
Assuntos
Miosina Tipo I , Miosinas , Actinas/metabolismo , Linfócitos B/metabolismo , Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal , Linfonodos/metabolismo , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Miosinas/genética , Miosinas/metabolismo , FosforilaçãoRESUMO
Myosins, a class of actin-based motor proteins existing in almost any organism, are originally considered only involved in driving muscle contraction, reshaping actin cytoskeleton, and anchoring or transporting cargoes, including protein complexes, organelles, vesicles. However, accumulating evidence reveals that myosins also play vital roles in viral infection, depending on viral species and infection stages. This review systemically summarizes the described various myosins, the performed functions, and the involved mechanisms or molecular pathways during viral infection. Meanwhile, the existing issues are also discussed. Additionally, the important technologies or agents, including siRNA, gene editing, and myosin inhibitors, would facilitate dissecting the actions and mechanisms for described and undescribed myosins, which could be adopted to prevent or control viral infection are also characterized.
Assuntos
Miosinas , Viroses , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Humanos , Miosinas/metabolismo , Organelas/metabolismo , Viroses/metabolismoRESUMO
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/metabolismo , Mutação de Sentido Incorreto/genética , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Sarcômeros/metabolismo , Adenosina Trifosfatases , Animais , Cardiomiopatia Hipertrófica/genética , Células Cultivadas , Metabolismo Energético , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Simulação de Dinâmica Molecular , Relaxamento Muscular , Contração Miocárdica , Miócitos Cardíacos/citologia , Conformação Proteica , Sarcômeros/genéticaRESUMO
OBJECTIVE: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). CONCLUSIONS: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.
Assuntos
Coagulação Sanguínea , Miosinas Cardíacas/metabolismo , Hemostasia , Trombina/biossíntese , Trombose/fisiopatologia , Animais , Plaquetas/metabolismo , Miosinas Cardíacas/fisiologia , Modelos Animais de Doenças , Fator Va/metabolismo , Fator Xa/metabolismo , Hemorragia/fisiopatologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Protrombina/metabolismoRESUMO
Neuronal dendrites are highly branched and specialized compartments with distinct structures and secretory organelles (e.g., spines, Golgi outposts), and a unique cytoskeletal organization that includes microtubules of mixed polarity. Dendritic membranes are enriched with proteins, which specialize in the formation and function of the post-synaptic membrane of the neuronal synapse. How these proteins partition preferentially in dendrites, and how they traffic in a manner that is spatiotemporally accurate and regulated by synaptic activity are long-standing questions of neuronal cell biology. Recent studies have shed new insights into the spatial control of dendritic membrane traffic, revealing new classes of proteins (e.g., septins) and cytoskeleton-based mechanisms with dendrite-specific functions. Here, we review these advances by revisiting the fundamental mechanisms that control membrane traffic at the levels of protein sorting and motor-driven transport on microtubules and actin filaments. Overall, dendrites possess unique mechanisms for the spatial control of membrane traffic, which might have specialized and co-evolved with their highly arborized morphology.
Assuntos
Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Neurônios/citologia , Transporte Proteico/fisiologia , Animais , Dendritos/metabolismo , Complexo de Golgi/metabolismo , HumanosRESUMO
Unlike the intermediate filament- and septin-based cytoskeletons which are apolar structures, the microtubule (MT) and actin cytoskeletons are polarized structures in mammalian cells and tissues including the testis, most notable in Sertoli cells. In the testis, these cytoskeletons that stretch across the epithelium of seminiferous tubules and lay perpendicular to the basement membrane of tunica propria serve as tracks for corresponding motor proteins to support cellular cargo transport. These cargoes include residual bodies, phagosomes, endocytic vesicles and most notably developing spermatocytes and haploid spermatids which lack the ultrastructures of motile cells (e.g., lamellipodia, filopodia). As such, these developing germ cells require the corresponding motor proteins to facilitate their transport across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Due to the polarized natures of these cytoskeletons with distinctive plus (+) and minus (-) end, directional cargo transport can take place based on the use of corresponding actin- or MT-based motor proteins. These include the MT-based minus (-) end directed motor proteins: dyneins, and the plus (+) end directed motor proteins: kinesins, as well as the actin-based motor proteins: myosins, many of which are plus (+) end directed but a few are also minus (-) end directed motor proteins. Recent studies have shown that these motor proteins are essential to support spermatogenesis. In this review, we briefly summarize and evaluate these recent findings so that this information will serve as a helpful guide for future studies and for planning functional experiments to better understand their role mechanistically in supporting spermatogenesis.
Assuntos
Dineínas , Espermatogênese , Animais , Masculino , Miosinas , Epitélio Seminífero , Células de Sertoli , EspermátidesRESUMO
Myosins play a key role in many cellular processes such as cell migration, adhesion, intracellular trafficking and internalization processes, making them ideal targets for bacteria. Through selected examples, such as enteropathogenic E. coli (EPEC), Neisseria, Salmonella, Shigella, Listeria or Chlamydia, this review aims to illustrate how bacteria target and hijack host cell myosins in order to adhere to the cell, to enter the cell by triggering their internalization, to evade from the cytosolic autonomous cell defense, to promote the biogenesis of intracellular replicative niche, to disseminate in tissues by cell-to-cell spreading, to exit out the host cell, and also to evade from macrophage phagocytosis. It highlights the diversity and sophistication of the strategy evolved by bacteria to manipulate one of their privileged targets, the actin cytoskeleton.
Assuntos
Infecções Bacterianas/metabolismo , Fenômenos Fisiológicos Bacterianos , Interações Hospedeiro-Patógeno , Miosinas/metabolismo , Animais , Bactérias/metabolismo , Aderência Bacteriana , Infecções Bacterianas/microbiologia , Citoplasma/metabolismo , Citoplasma/microbiologia , HumanosRESUMO
While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical-dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology and identity. Our analysis across timescales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule-based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway.
RESUMO
Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule-organizing center in a "cloud," and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biological functions, such as cargo degradation, antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biological events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid-protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the molecular machineries that control this mobility.