Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.187
Filtrar
1.
PLoS Biol ; 18(11): e3000925, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33216759

RESUMO

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.


Assuntos
Actinas/química , Proteínas dos Microfilamentos/química , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Toxinas Bacterianas/química , Sítios de Ligação , Ligação Competitiva , Cofilina 1/química , Cofilina 1/ultraestrutura , Microscopia Crioeletrônica , Corantes Fluorescentes/química , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Confocal , Modelos Moleculares , Miosinas/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura
2.
Nat Commun ; 11(1): 5897, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214556

RESUMO

Since the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.


Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Difosfato de Adenosina/metabolismo , Fosfatos/metabolismo , Pirenos/química , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Microscopia Crioeletrônica , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Iodoacetamida/análogos & derivados , Iodoacetamida/química , Cinética , Proteínas dos Microfilamentos/metabolismo , Polimerização , Ligação Proteica
3.
Arch Biochem Biophys ; 695: 108624, 2020 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-33049292

RESUMO

Mutations in the α-cardiac actin ACTC1 gene cause dilated or hypertrophic cardiomyopathy. These diseases are the result of changes in protein interactions between ACTC protein and force-generating ß-myosin or the calcium-dependent cardiac-tropomyosin (cTm) and cardiac troponin (cTn) regulatory complex, altering the overall contractile force. The T126I and S271F ACTC variants possess amino acid substitutions on the other side of actin relative to the myosin or regulatory protein binding sites on what we call the "dark side" of actin. The T126I change results in hyposensitivity to calcium, in accordance with the calcium sensitivity pathway of cardiomyopathy development while the S271F change alters the maximum in vitro motility sliding speed, reflecting a change in maximum force. These results demonstrate the role of actin allostery in the cardiac disease development.


Assuntos
Actinas/química , Cardiomiopatias , Actinas/genética , Actinas/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Animais , Humanos , Mutação de Sentido Incorreto , Células Sf9 , Spodoptera , Troponina/química , Troponina/genética , Troponina/metabolismo
4.
PLoS Biol ; 18(9): e3000848, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898131

RESUMO

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Miocárdio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Sarcômeros/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(40): 24670-24678, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958682

RESUMO

Cell crawling requires the generation of intracellular forces by the cytoskeleton and their transmission to an extracellular substrate through specific adhesion molecules. Crawling cells show many features of excitable systems, such as spontaneous symmetry breaking and crawling in the absence of external cues, and periodic and propagating waves of activity. Mechanical instabilities in the active cytoskeleton network and feedback loops in the biochemical network of activators and repressors of cytoskeleton dynamics have been invoked to explain these dynamical features. Here, I show that the interplay between the dynamics of cell-substrate adhesion and linear cellular mechanics is sufficient to reproduce many nonlinear dynamical patterns observed in spreading and crawling cells. Using an analytical formalism of the molecular clutch model of cell adhesion, regulated by local mechanical forces, I show that cellular traction forces exhibit stick-slip dynamics resulting in periodic waves of protrusion/retraction and propagating waves along the cell edge. This can explain spontaneous symmetry breaking and polarization of spreading cells, leading to steady crawling or bipedal motion, and bistability, where persistent cell motion requires a sufficiently strong transient external stimulus. The model also highlights the role of membrane tension in providing the long-range mechanical communication across the cell required for symmetry breaking.


Assuntos
Actinas/metabolismo , Polaridade Celular , Células/citologia , Actinas/química , Fenômenos Biomecânicos , Adesão Celular , Movimento Celular , Extensões da Superfície Celular , Células/química , Células/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Cinética , Modelos Biológicos
6.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
7.
Oncogene ; 39(38): 6085-6098, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32801337

RESUMO

The mechanistic basis of liver metastasis in colorectal cancer remains poorly understood. We previously reported that the sclerostin domain containing-1 (SOSTDC1) protein is overexpressed in the secretome of metastatic colorectal cancer cells and can inhibit liver homing. Here, we investigated the mechanisms of SOSTDC1 for promoting invasiveness and progression of colorectal cancer liver metastasis. SOSTDC1 inhibition of BMP4 maintains the expression of cancer stem cell traits, including SOX2 and NANOG. Immunoprecipitation and mass spectrometry analyses reveal the association of SOSTDC1 with ALCAM/CD166, which was confirmed by confocal microscopy and competition ELISA. Interaction with ALCAM is mediated by the N-terminal region of SOSTDC1, which contains a sequence similar to the ALCAM-binding motif used by CD6. Knocking down either SOSTDC1 or ALCAM expression, or using blocking antibodies, reduces the invasive activity by inhibiting Src and PI3K/AKT signaling pathways. In addition, ALCAM interacts with the α2ß1 and α1ß1 integrins, providing a possible link to Src activation. Finally, inoculation of SOSTDC1-silenced metastatic cells increases mouse survival by inhibiting liver metastasis. In conclusion, SOSTDC1 promotes invasion and liver metastasis in colorectal cancer, by overcoming BMP4-specific antimetastatic signals and inducing ALCAM-mediated Src and PI3K/AKT activation. These experiments underscore the potential of SOSTDC1 as a therapeutic target in metastatic colorectal cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Fetais/metabolismo , Neoplasias Hepáticas/secundário , Actinas/química , Actinas/metabolismo , Animais , Biomarcadores Tumorais , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(36): 22128-22134, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848053

RESUMO

Dendritic spines are tiny membranous protrusions on the dendrites of neurons. Dendritic spines change shape in response to input signals, thereby strengthening the connections between neurons. The growth and stabilization of dendritic spines is thought to be essential for maintaining long-term memory. Actin cytoskeleton remodeling in spines is a key element of their formation and growth. More speculatively, the aggregation of CPEB3, a functional prion that binds RNA, has been reported to be involved in the maintenance of long-term memory. Here we study the interaction between actin and CPEB3 and propose a molecular model for the complex structure of CPEB3 and an actin filament (F-actin). The results of our computational modeling, including both energetic and structural analyses, are compared with novel data from peptide array experiments. Our model of the CPEB3/F-actin interaction suggests that F-actin potentially triggers the aggregation-prone structural transition of a short CPEB3 sequence by zipping it into a beta-hairpin form. We also propose that the CPEB3/F-actin interaction might be regulated by the SUMOylation of CPEB3, based on bioinformatic searches for potential SUMOylation sites as well as SUMO interacting motifs in CPEB3. On the basis of these results and the existing literature, we put forward a possible molecular mechanism underlying long-term memory that involves CPEB3's binding to actin, its aggregation, and its regulation by SUMOylation.


Assuntos
Actinas/química , Proteínas de Ligação a RNA/química , Actinas/metabolismo , Motivos de Aminoácidos , Simulação por Computador , Humanos , Memória de Longo Prazo , Modelos Moleculares , Neurônios/química , Neurônios/fisiologia , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , Sumoilação
9.
Proc Natl Acad Sci U S A ; 117(33): 19904-19913, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747565

RESUMO

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Gelsolina/metabolismo , Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/genética , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Evolução Molecular , Gelsolina/química , Gelsolina/genética , Genoma Arqueal , Polimerização , Conformação Proteica em alfa-Hélice , Alinhamento de Sequência
10.
Proc Natl Acad Sci U S A ; 117(31): 18822-18831, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690703

RESUMO

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.


Assuntos
Cardiomiopatias/metabolismo , Mutação/genética , Tropomiosina , Troponina T , Actinas/química , Actinas/metabolismo , Animais , Cálcio/metabolismo , Diástole/genética , Diástole/fisiologia , Proteínas de Drosophila , Humanos , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Ligação Proteica , Ratos , Tropomiosina/química , Tropomiosina/metabolismo , Troponina T/química , Troponina T/genética , Troponina T/metabolismo
11.
PLoS Comput Biol ; 16(7): e1007972, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658891

RESUMO

Although quantitative insights into the killing behaviour of Cytotoxic T Lymphocytes (CTLs) are necessary for the rational design of immune-based therapies, CTL killing function remains insufficiently characterised. One established model of CTL killing treats CTL cytotoxicity as a Poisson process, based on the assumption that CTLs serially kill antigen-presenting target cells via delivery of lethal hits, each lethal hit corresponding to a single injection of cytotoxic proteins into the target cell cytoplasm. Contradicting this model, a recent in vitro study of individual CTLs killing targets over a 12-hour period found significantly greater heterogeneity in CTL killing performance than predicted by Poisson-based killing. The observed killing process was dynamic and varied between CTLs, with the best performing CTLs exhibiting a marked increase in killing during the final hours of the experiments, along with a "burst killing" kinetic. Despite a search for potential differences between CTLs, no mechanistic explanation for the heterogeneous killing kinetics was found. Here we have used stochastic simulations to assess whether target cells might require multiple hits from CTLs before undergoing apoptosis, in order to verify whether multiple-hitting could explain the late onset, burst killing dynamics observed in vitro. We found that multiple-hitting from CTLs was entirely consistent with the observed killing kinetics. Moreover, the number of available targets and the spatiotemporal kinetics of CTL:target interactions influenced the realised CTL killing rate. We subsequently used realistic, spatial simulations to assess methods for estimating the hitting rate and the number of hits required for target death, to be applied to microscopy data of individual CTLs killing targets. We found that measuring the cumulative duration of individual contacts that targets have with CTLs would substantially improve accuracy when estimating the killing kinetics of CTLs.


Assuntos
Citoplasma/metabolismo , Processos Estocásticos , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Actinas/química , Algoritmos , Células Apresentadoras de Antígenos , Apoptose , Movimento Celular , Simulação por Computador , Citotoxicidade Imunológica , Humanos , Cinética , Funções Verossimilhança , Método de Monte Carlo , Distribuição de Poisson
12.
Proc Natl Acad Sci U S A ; 117(31): 18511-18520, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690698

RESUMO

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.


Assuntos
Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Divisão Celular , Chlamydomonas/química , Citocinese , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Ligação Proteica
13.
Exp Parasitol ; 217: 107956, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32659234

RESUMO

The species name Cryptosporidium bollandi n. sp. is proposed for Cryptosporidium piscine genotype 2 based on morphological, biological and molecular characterisation. Phylogenetic analyses of 18S rRNA (18S) sequences revealed that C. bollandi n. sp. was most closely related to piscine genotype 4 (5.1% genetic distance) and exhibited genetic distances of 10.0%, 12.2% and 25.2% from Cryptosporidium molnari, Cryptosporidium huwi and Cryptosporidium scophthtalmi, respectively. At the actin locus, C. bollandi n. sp. was again most closely related to piscine genotype 4 (6.8% genetic distance) and exhibited 15.5% (C. molnari), 18.4% (C. huwi), 22.9% (C. scophthalmi) and up to 27.5% genetic distance from other Cryptosporidium spp. (Cryptosporidium felis). Phylogenetic analysis of concatenated 18S and actin sequences showed that C. bollandi n. sp. exhibited 12.9% (C. molnari) to 21.1% (C. canis) genetic distance from all other Cryptosporidium spp. Genetic data as well as previous histological analysis clearly supports the validity of C. bollandi n. sp. as a separate species.


Assuntos
Ciclídeos/parasitologia , Criptosporidiose/parasitologia , Cryptosporidium/fisiologia , Doenças dos Peixes/parasitologia , Actinas/química , Actinas/genética , Animais , Sequência de Bases , Evolução Biológica , Criptosporidiose/epidemiologia , Cryptosporidium/classificação , Cryptosporidium/genética , Cryptosporidium/ultraestrutura , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças dos Peixes/epidemiologia , Pesqueiros , Genótipo , Funções Verossimilhança , Microscopia Eletrônica de Transmissão/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Ribossômico 18S/química , Washington/epidemiologia , Austrália Ocidental/epidemiologia
14.
Science ; 368(6496): 1205-1210, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527825

RESUMO

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.


Assuntos
Actinas/metabolismo , Movimento Celular , Extensões da Superfície Celular , Actinas/química , Actinas/genética , Membrana Celular , Polaridade Celular , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos
15.
F1000Res ; 9: 52, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-318745

RESUMO

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has expanded the number of highly pathogenic coronaviruses affecting humans. The 2019-nCoV represents a potential epidemic or pandemic threat, which requires a quick response for preparedness against this infection. The present report uses the informational spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. The performed in silico analysis indicates that the newly emerging 2019-nCoV is closely related to severe acute respiratory syndrome (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) might be a putative receptor for the novel virus as well. Additional results indicated that civets and poultry are potential candidates for the natural reservoir of the 2019-nCoV, and that domain 288-330 of S1 protein from the 2019-nCoV represents promising therapeutic and/or vaccine target.


Assuntos
Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Tropismo Viral , Actinas/química , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , Humanos , Ibuprofeno/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Receptores Virais/química , Vírus da SARS
16.
F1000Res ; 9: 52, 2020.
Artigo em Inglês | MEDLINE | ID: covidwho-400221

RESUMO

A novel coronavirus recently identified in Wuhan, China (2019-nCoV) has expanded the number of highly pathogenic coronaviruses affecting humans. The 2019-nCoV represents a potential epidemic or pandemic threat, which requires a quick response for preparedness against this infection. The present report uses the informational spectrum methodology to identify the possible origin and natural host of the new virus, as well as putative therapeutic and vaccine targets. The performed in silico analysis indicates that the newly emerging 2019-nCoV is closely related to severe acute respiratory syndrome (SARS)-CoV and, to a lesser degree, Middle East respiratory syndrome (MERS)-CoV. Moreover, the well-known SARS-CoV receptor (ACE2) might be a putative receptor for the novel virus as well. Additional results indicated that civets and poultry are potential candidates for the natural reservoir of the 2019-nCoV, and that domain 288-330 of S1 protein from the 2019-nCoV represents promising therapeutic and/or vaccine target.


Assuntos
Betacoronavirus/química , Glicoproteína da Espícula de Coronavírus/química , Tropismo Viral , Actinas/química , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , Humanos , Ibuprofeno/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio , Pandemias , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Receptores Virais/química , Vírus da SARS
17.
Adv Exp Med Biol ; 1239: 7-19, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451853

RESUMO

Directed movements on actin filaments within the cell are powered by molecular motors of the myosin superfamily. On actin filaments, myosin motors convert the energy from ATP into force and movement. Myosin motors power such diverse cellular functions as cytokinesis, membrane trafficking, organelle movements, and cellular migration. Myosin generates force and movement via a number of structural changes associated with hydrolysis of ATP, binding to actin, and release of the ATP hydrolysis products while bound to actin. Herein we provide an overview of those structural changes and how they relate to the actin-myosin ATPase cycle. These structural changes are the basis of chemo-mechanical transduction by myosin motors.


Assuntos
Miosinas/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Movimento , Miosinas/metabolismo
18.
Adv Exp Med Biol ; 1239: 41-59, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451855

RESUMO

After several decades studying different acto-myosin complexes at lower and intermediate resolution - limited by the electron microscope instrumentation available then - recent advances in imaging technology have been crucial for obtaining a number of excellent high-resolution 3D reconstructions from cryo electron microscopy. The resolution level reached now is about 3-4 Å, which allows unambiguous model building of filamentous actin on its own as well as that of actin filaments decorated with strongly bound myosin variants. The interface between actin and the myosin motor domain can now be described in detail, and the function of parts of the interface (such as, e.g., the cardiomyopathy loop) can be understood in a mechanistical way. Most recently, reconstructions of actin filaments decorated with different myosins, which show a strongly bound acto-myosin complex also in the presence of the nucleotide ADP, have become available. The comparison of these structures with the nucleotide-free Rigor state provide the first mechanistic description of force sensing. An open question is still the initial interaction of the motor domain of myosin with the actin filament. Such weakly interacting states have so far not been the subject of microscopical studies, even though high-resolution structures would be needed to shed light on the initial steps of phosphate release and power stroke initiation.


Assuntos
Actomiosina/química , Citoesqueleto de Actina , Actinas/química , Actomiosina/ultraestrutura , Microscopia Crioeletrônica , Miosinas/química
19.
Nat Commun ; 11(1): 2417, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415060

RESUMO

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/química , Doenças Musculares/patologia , Animais , Animais Geneticamente Modificados , Cardiomiopatia Hipertrófica , Biologia Computacional , Drosophila melanogaster/metabolismo , Feminino , Voo Animal , Humanos , Ligação de Hidrogênio , Masculino , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Contração Muscular , Mutação , Análise de Componente Principal , Multimerização Proteica , Eletricidade Estática , Transgenes , Tropomiosina/química
20.
Sci Rep ; 10(1): 8133, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424215

RESUMO

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.


Assuntos
Macrófagos/química , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Actinas/química , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Fluorescência , Células HeLa , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Fagocitose , Células RAW 264.7
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA