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Actin filament assembly and the regulation of its mechanical properties are fundamental processes essential for eukaryotic cell function. Residue E167 in vertebrate actins forms an inter-subunit salt bridge with residue K61 of the adjacent subunit. Saccharomyces cerevisiae actin filaments are more flexible than vertebrate filaments and have an alanine at this position (A167). Substitution of this alanine for a glutamic acid (A167E) confers Saccharomyces cerevisiae actin filaments with salt-dependent stiffness similar to vertebrate actins. We developed an optimized cryogenic electron microscopy workflow refining sample preparation and vitrification to obtain near-atomic resolution structures of wild-type and A167E mutant Saccharomyces cerevisiae actin filaments. The difference between these structures allowed us to pinpoint the potential binding site of a filament-associated cation that controls the stiffness of the filaments in vertebrate and A167E Saccharomyces cerevisiae actins. Through an analysis of previously published high-resolution reconstructions of vertebrate actin filaments, along with a newly determined high-resolution vertebrate actin structure in the absence of potassium, we identified a unique peak near residue 167 consistent with the binding of a magnesium ion. Our findings show how magnesium can contribute to filament stiffening by directly bridging actin subunits and allosterically affecting the orientation of the DNase-I binding loop of actin, which plays a regulatory role in modulating actin filament stiffness and interactions with regulatory proteins.
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Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.
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Fenotipo , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Pirofosfatasas/genética , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Femenino , Variación Genética , Masculino , Mutación/genéticaRESUMEN
Municipal solid waste incineration fly ash (IFA) designated as hazardous waste poses risks to environment and human health. This study introduces a novel approach for the stabilization and solidification (S/S) of IFA: a combined approach involving alkali treatment and immobilization in low-carbon supersulfated cement (SSC). The impact of varying temperatures of alkali solution on the chemical and mineralogical compositions, as well as the pozzolanic reactivity of IFA, and the removal efficiency of heavy metals and metallic aluminum (Al) were examined. The physical characteristics, hydration kinetics and effectiveness of SSC in immobilizing IFA were also analyzed. Results showed that alkali treatment at 25 °C effectively eliminated heavy metals like manganese (Mn), barium (Ba), nickel (Ni), and chromium (Cr) to safe levels and totally removed the metallic Al, while enhancing the pozzolanic reactivity of IFA. By incorporating the alkali-treated IFA and filtrate, the density, compressive strength and hydration reaction of SSC were improved, resulting in higher hydration degree, finer pore structure, and denser microstructure compared to untreated IFA. The rich presence of calcium-aluminosilicate-hydrate (C-(A)-S-H) and ettringite (AFt) in SSC facilitated the efficient stabilization and solidification of heavy metals, leading to a significant decrease in their leaching potential. The use of SSC for treating Ca(OH)2- and 25°C-treated IFA could achieve high strength and high-efficient immobilization.
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Álcalis , Ceniza del Carbón , Materiales de Construcción , Incineración , Metales Pesados , Residuos Sólidos , Ceniza del Carbón/química , Metales Pesados/química , Álcalis/química , Eliminación de Residuos/métodos , Aluminio/químicaRESUMEN
[This corrects the article DOI: 10.2196/50561.].
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High uncertainty in optical properties of black carbon (BC) involving heterogeneous chemistry has recently attracted increasing attention in the field of atmospheric climatology. To fill the gap in BC optical knowledge so as to estimate more accurate climate effects and serve the response to global warming, it is beneficial to conduct site-level studies on BC light absorption enhancement (Eabs) characteristics. Real-time surface gas and particulate pollutant observations during the summer and winter over Wuhan were utilized for the analysis of Eabs simulated by minimum R squared (MRS), considering two distinct atmospheric conditions (2015 and 2017). In general, differences in aerosol emissions led to Eabs differential behaviors. The summer average of Eabs (1.92 ± 0.55) in 2015 was higher than the winter average (1.27 ± 0.42), while the average (1.11 ± 0.20) in 2017 summer was lower than that (1.67 ± 0.69) in winter. Eabs and RBC (representing the mass ratio of non-refractory constituents to elemental carbon) constraints suggest that Eabs increased with the increase in RBC under the ambient condition enriched by secondary inorganic aerosol (SIA), with a maximum growth rate of 70.6% in 2015 summer. However, Eabs demonstrated a negative trend against RBC in 2017 winter due to the more complicated mixing state. The result arose from the opposite impact of hygroscopic SIA and absorbing OC/irregular distributed coatings on amplifying the light absorbency of BC. Furthermore, sensitivity analysis revealed a robust positive correlation (R > 0.9) between aerosol chemical compositions (including sulfate, nitrate, ammonium and secondary organic carbon), which could be significantly perturbed by only a small fraction of absorbing materials or restructuring BC through gaps filling. The above findings not only deepen the understanding of BC, but also provide useful information for the scientific decision-making in government to mitigate particulate pollution and obtain more precise BC radiative forcing.
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Aerosoles , Contaminantes Atmosféricos , Monitoreo del Ambiente , Hollín , Contaminantes Atmosféricos/análisis , Aerosoles/análisis , Monitoreo del Ambiente/métodos , Estaciones del Año , Material Particulado/análisis , Luz , Carbono , China , Atmósfera/químicaRESUMEN
Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeFx bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeFx transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of Pi from the ADP-Pi transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.
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Citoesqueleto de Actina , Fosfatos , Microscopía por Crioelectrón , Citoesqueleto , Actinas , Complejo 2-3 Proteico Relacionado con la ActinaRESUMEN
Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.
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Actinas , Cofilina 1 , Humanos , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Quinasas Lim/metabolismo , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: Tumor immunotherapy is an innovative treatment today, but there are limited data on the quality of immunotherapy information on social networks. Dissemination of misinformation through the internet is a major social issue. OBJECTIVE: Our objective was to characterize the quality of information and presence of misinformation about tumor immunotherapy on internet-based videos commonly used by the Chinese population. METHODS: Using the keyword "tumor immunotherapy" in Chinese, we searched TikTok, Tencent, iQIYI, and BiliBili on March 5, 2022. We reviewed the 118 screened videos using the Patient Education Materials Assessment Tool-a validated instrument to collect consumer health information. DISCERN quality criteria and the JAMA (Journal of the American Medical Association) Benchmark Criteria were used for assessing the quality and reliability of the health information. The videos' content was also evaluated. RESULTS: The 118 videos about tumor immunotherapy were mostly uploaded by channels dedicated to lectures, health-related animations, and interviews; their median length was 5 minutes, and 79% of them were published in and after 2018. The median understandability and actionability of the videos were 71% and 71%, respectively. However, the quality of information was moderate to poor on the validated DISCERN and JAMA assessments. Only 12 videos contained misinformation (score of >1 out of 5). Videos with a doctor (lectures and interviews) not only were significantly less likely to contain misinformation but also had better quality and a greater forwarding number. Moreover, the results showed that more than half of the videos contain little or no content on the risk factors and management of tumor immunotherapy. Overall, over half of the videos had some or more information on the definition, symptoms, evaluation, and outcomes of tumor immunotherapy. CONCLUSIONS: Although the quality of immunotherapy information on internet-based videos commonly used by Chinese people is moderate, these videos have less misinformation and better content. Caution must be exercised when using these videos as a source of tumor immunotherapy-related information.
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The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.
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Factores Despolimerizantes de la Actina , Actinas , Actinas/metabolismo , Actomiosina/metabolismo , Actinina , Citoesqueleto de Actina/metabolismo , MiosinasRESUMEN
Cooperative ligand binding to linear polymers is fundamental in many scientific disciplines, particularly biological and chemical physics and engineering. Such ligand binding interactions have been widely modeled using infinite one-dimensional (1D) Ising models even in cases where the linear polymers are more complex (e.g. actin filaments and other double-stranded linear polymers). Here, we use sequence-generating and transfer matrix methods to obtain an analytical method for cooperative equilibrium ligand binding to double-stranded Ising lattices. We use this exact solution to evaluate binding properties and features and analyze experimental binding data of cooperative binding of the regulatory protein, cofilin, to actin filaments. This analysis, with additional experimental information about the observed bound cofilin cluster sizes and filament structure, reveals that a bound cofilin promotes cooperative binding to its longitudinal nearest-neighbors but has very modest effects on lateral nearest-neighbors. The bound cofilin cluster sizes calculated from the best fit parameters from the double-stranded model are considerably larger than when calculated with the 1D model, consistent with experimental observations made by electron microscopy and fluorescence imaging. The exact solution obtained and the method for using the solution developed here can be widely used for analysis of variety of multistranded lattice systems.
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The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation.
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Actinas , Actomiosina , Fricción , Contracción Muscular , Membrana Dobles de LípidosRESUMEN
Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but the aspects of cofilin functionality driving this conservation are not clear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and subsequent biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. While the presence of a serine, rather than threonine, phosphoacceptor residue was essential for phosphorylation by LIM kinase, the native cofilin N-terminus was otherwise a suboptimal LIM kinase substrate. This circumstance was not due to sequence requirements for actin binding and severing, but rather appeared primarily to maintain the capacity for phosphorylation to inactivate cofilin. Overall, the individual sequence requirements for cofilin function and regulation were remarkably loose when examined separately, but collectively restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a regulatory phosphorylation site can balance potentially competing sequence requirements for function and regulation.
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Objective: The influence of continuous renal replacement therapy (CRRT) on the steady-state plasma concentration of high-dose tigecycline was investigated in septic shock patients to provide references for drug dosing. Methods: In this prospective observational study, 17 septic shock patients presenting with severe infections needing a broad-spectrum antibiotic therapy with high-dose tigecycline (100 mg per 12 h) in the intensive care unit were included and divided into CRRT group (n = 6) or non-CRRT group (n = 11). The blood samples were collected and plasma drug concentration was determined by SHIMADZU LC-20A and SHIMADZU LCMS 8040. The steady-state plasma concentration was compared between groups using unpaired t-test. Furthermore, between-groups comparisons adjusted for baseline value was also done using multivariate linear regression model. Results: Peak concentration (Cmax) of tigecycline was increased in CRRT group compared to non-CRRT group, but there were no statistical differences (505.11 ± 143.84 vs. 406.29 ± 108.00 ng/mL, p-value: 0.129). Trough concentration (Cmin) of tigecycline was significantly higher in CRRT group than in non-CRRT group, with statistical differences (287.92 ± 41.91 vs. 174.79 ± 33.15 ng/mL, p-value: 0.000, adjusted p-value: 0.000). In safety, Cmin was reported to be a useful predictor of hepatotoxicity with a cut-off of 474.8 ng/mL. In our studies, Cmin of all patients in CRRT group was lower than 474.8 ng/mL. Conclusion: The plasma concentration of tigecycline was increased in septic shock patients with CRRT treatment and only Cmin shown statistical differences. No dose adjustment seems needed in the view of hepatotoxicity. Clinical Trial Registration: https://www.chictr.org.cn/, identifier ChiCTR2000037475.
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Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.
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Citoesqueleto de Actina , Citoesqueleto , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Miosinas/metabolismo , Unión Proteica , Actinas/metabolismoRESUMEN
Smooth muscle cell (SMC) phenotypic switch from a quiescent 'contractile' phenotype to a dedifferentiated and proliferative state underlies the development of cardiovascular diseases (CVDs); however, our understanding of the mechanism is still incomplete. In the present study, we explored the potential role of ferroptosis, a novel nonapoptotic form of cell death, in SMC phenotypic switch and related neointimal formation. We found that ferroptotic stress was triggered in cultured dedifferentiated SMCs and arterial neointimal tissue of wire-injured mice. Moreover, pro-ferroptosis stress was activated in arterial neointimal tissue of clinical patients who underwent carotid endarterectomy. Blockade of ferroptotic stress via administration of a pharmacological inhibitor or by global genetic overexpression of glutathione peroxidase-4 (GPX4), a well-established anti-ferroptosis molecule, delayed SMC phenotype switch and arterial remodelling. Conditional SMC-specific gene delivery of GPX4 using adreno-associated virus in the carotid artery inhibited ferroptosis and prevented neointimal formation. Conversely, ferroptosis stress directly triggered dedifferentiation of SMCs. Transcriptomics analysis demonstrated that inhibition of ferroptotic stress mainly targets the mitochondrial respiratory chain and oxidative phosphorylation. Mechanistically, ferroptosis inhibition corrected the disrupted mitochondrial homeostasis in dedifferentiated SMCs, including enhanced mitochondrial ROS production, dysregulated mitochondrial dynamics, and mitochondrial hyperpolarization, and ultimately inhibited SMC phenotypic switch and growth. Copper-diacetyl-bisN4-methylthiosemicarbazone (CuATSM), an agent used for clinical molecular imaging and that potently inhibits ferroptosis, prevented SMC phenotypic switch, neointimal formation and arterial inflammation in mice. These results indicate that pro-ferroptosis stress is likely to promote SMC phenotypic switch during neointimal formation and imply that inhibition of ferroptotic stress may be a promising translational approach to treat CVDs with SMC phenotype switch.
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Desdiferenciación Celular , Miocitos del Músculo Liso , Ratones , Animales , Células Cultivadas , Homeostasis , Miocitos del Músculo Liso/metabolismo , Músculo Liso , Proliferación CelularRESUMEN
The question of how to mine safely in close multi coal seams is the main concern for coal operators, in particular for large-dip coal seams with complex geological and mechanical conditions. This paper presents a detailed similarity simulation on the movement characteristics of the overburden and the stress distribution of underlying strata in terms of a specific coal mine in the Tielieke mining area of the Kubai coalfield via a three-dimensional photogrammetry system and a high-speed static resistance analyzer. The results show that the overburden strata are asymmetrically deformed around the coal pillar and the fracture area is perpendicular to the longwall with an "M" shape when deeper coal is mined. Moreover, the asymmetric movement of overburden results in the non-uniform distribution of stress on the floor of the coal pillar and ribs. In particular, stress is closely related to the location of the longwall, and stress of the coal pillar is much larger when it is closer to the deep side. The floor stress relief degree of the longwall in the deep zone is higher than that of its counterparts, providing a theoretical foundation for a reasonable layout and a support technique for roadways. The main contribution of this research that it can be used as a reference in maintaining the integrity of surrounding rock for large-dip coal seams with close distances.
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Minas de Carbón , Carbón Mineral , Simulación por Computador , Geología , Modelos TeóricosRESUMEN
The DEAD-box protein Dbp5 is essential for RNA export, which involves regulation by the nucleoporins Gle1 and Nup159 at the cytoplasmic face of the nuclear pore complex (NPC). Mechanistic understanding of how these nucleoporins regulate RNA export requires analyses of the intrinsic and activated Dbp5 ATPase cycle. Here, kinetic and equilibrium analyses of the Saccharomyces cerevisiae Gle1-activated Dbp5 ATPase cycle are presented, indicating that Gle1 and ATP, but not ADP-Pi or ADP, binding to Dbp5 are thermodynamically coupled. As a result, Gle1 binds Dbp5-ATP > 100-fold more tightly than Dbp5 in other nucleotide states and Gle1 equilibrium binding of ATP to Dbp5 increases >150-fold via slowed ATP dissociation. Second, Gle1 accelerated Dbp5 ATPase activity by increasing the rate-limiting Pi release rate constant â¼20-fold, which remains rate limiting. These data show that Gle1 activates Dbp5 by modulating ATP binding and Pi release. These Gle1 activities are expected to facilitate ATPase cycling, ensuring a pool of ATP bound Dbp5 at NPCs to engage RNA during export. This work provides a mechanism of Gle1-activation of Dbp5 and a framework to understand the joint roles of Gle1, Nup159, and other nucleoporins in regulating Dbp5 to mediate RNA export and other Dbp5 functions in gene expression.
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ARN Helicasas DEAD-box , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfatos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Wave-like beating of eukaryotic cilia and flagella-threadlike protrusions found in many cells and microorganisms-is a classic example of spontaneous mechanical oscillations in biology. This type of self-organized active matter raises the question of the coordination mechanism between molecular motor activity and cytoskeletal filament bending. Here we show that in the presence of myosin motors, polymerizing actin filaments self-assemble into polar bundles that exhibit wave-like beating. Importantly, filament beating is associated with myosin density waves initiated at twice the frequency of the actin-bending waves. A theoretical description based on curvature control of motor binding to the filaments and of motor activity explains our observations in a regime of high internal friction. Overall, our results indicate that the binding of myosin to actin depends on the actin bundle shape, providing a feedback mechanism between the myosin activity and filament deformations for the self-organization of large motor filament assemblies.
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Members of the ADF/cofilin family of regulatory proteins bind actin filaments cooperatively, locally change actin subunit conformation and orientation, and sever filaments at "boundaries" between bare and cofilin-occupied segments. A cluster of bound cofilin introduces two distinct classes of boundaries due to the intrinsic polarity of actin filaments, one at the "pointed" end side and the other at the "barbed" end-side of the cluster; severing occurs more readily at the pointed end side of the cluster ("fast-severing" boundary) than the barbed end side ("slow-severing" boundary). A recent electron-cryomicroscopy (cryo-EM) model of the slow-severing boundary revealed structural "defects" at the interface that potentially contribute to severing. However, the structure of the fast-severing boundary remains uncertain. Here, we use extensive molecular dynamics simulations to produce atomic resolution models of both severing boundaries. Our equilibrated simulation model of the slow-severing boundary is consistent with the cryo-EM structural model. Simulations indicate that actin subunits at both boundaries adopt structures intermediate between those of bare and cofilin-bound actin subunits. These "intermediate" states have compromised intersubunit contacts, but those at the slow-severing boundary are stabilized by cofilin bridging interactions, accounting for its lower fragmentation probability. Simulations where cofilin proteins are removed from cofilactin filaments favor a mechanism in which a cluster of two contiguously bound cofilins is needed to fully stabilize the cofilactin conformation, promote cooperative binding interactions, and accelerate filament severing. Together, these studies provide a molecular-scale foundation for developing coarse-grained and theoretical descriptions of cofilin-mediated actin filament severing.