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Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
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Células Cromafins , Dinamina II , Endocitose , Exocitose , Mutação , Miopatias Congênitas Estruturais , Células Cromafins/metabolismo , Endocitose/fisiologia , Endocitose/genética , Dinamina II/genética , Dinamina II/metabolismo , Animais , Exocitose/fisiologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/metabolismo , Mutação/genética , Bovinos , Humanos , Actinas/metabolismo , Actinas/genética , Células Cultivadas , Técnicas de Patch-Clamp , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologiaRESUMO
This study sought to assess how post-game creatine kinase (CK) levels correlate with the number of sprints and the impact of the ACTN3 polymorphism on this response. This research constituted a descriptive/observational, retrospective cross-sectional study. DNA was extracted from blood samples for ACTN3 polymorphism genotyping. CK was measured 48 h after official matches, and the number of sprints (>19 km/h) was tracked using Global Positioning System (GPS) technology. The main cohort included 23 professional soccer players from the top tier of the Brazilian Championship. We analyzed 115 GPS + CK data sets. The replication cohort comprised 18 professional soccer players from the First Division of the Championship, had the same methodology applied, and featured a total of 90 GPS (sprints > 25.2 km/h) + CK data sets. For the main cohort, a significant positive correlation was seen between the number of sprints and the CK levels (p = 0.009). Athletes with the ACTN3 RR genotype had higher CK levels as more sprints were performed during the match (p = 0.017). However, the relationship was not found for X allele carriers (p > 0.05). For the replication cohort, there was a near-significant correlation between CK levels and the number of sprints (p = 0.05), and RR individuals showed a significant association (p = 0.01), whereas X allele carriers did not (p = 0.06). A greater number of sprints during matches is linked to higher CK levels, primarily among players with the ACTN3 RR genotype, which is potentially due to an increased presence of type II muscle fibers. These findings were replicated for both cohorts of elite Brazilian soccer players, emphasizing the importance of genetic factors in injury prevention.
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Actinina , Creatina Quinase , Corrida , Futebol , Humanos , Actinina/genética , Brasil , Masculino , Creatina Quinase/sangue , Creatina Quinase/genética , Adulto , Atletas , Desempenho Atlético , Estudos Transversais , Estudos Retrospectivos , Genótipo , Polimorfismo de Nucleotídeo Único , Adulto Jovem , Polimorfismo GenéticoRESUMO
INTRODUCTION: The phenotypic consequences of the p.Arg577Ter variant in the α-actinin-3 (ACTN3) gene are suggestive of a trade-off between performance traits for speed and endurance sports. Although there is a consistent association of the c.1729C allele (aka R allele) with strength/power traits, there is still a debate on whether the null allele (c.1729T allele; aka X allele) influences endurance performance. The present study aimed to test the association of the ACTN3 p.Arg577Ter variant with long-distance endurance athlete status, using previously published data with the Brazilian population. METHODS: Genotypic data from 203 long-distance athletes and 1724 controls were analysed in a case-control approach. RESULTS: The frequency of the X allele was significantly higher in long-distance athletes than in the control group (51.5% vs. 41.4%; p = 0.000095). The R/X and X/X genotypes were overrepresented in the athlete group. Individuals with the R/X genotype instead of the R/R genotype had a 1.6 increase in the odds of being a long-distance athlete (p = 0.012), whereas individuals with the X/X genotype instead of the R/R genotype had a 2.2 increase in the odds of being a long-distance athlete (p = 0.00017). CONCLUSION: The X allele, mainly the X/X genotype, was associated with long-distance athlete status in Brazilians.
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Actinina , Alelos , Atletas , Humanos , Brasil , Actinina/genética , Masculino , Feminino , Adulto , Estudos Retrospectivos , Estudos de Casos e Controles , Genótipo , Frequência do Gene , Resistência Física/genética , Adulto Jovem , AdolescenteRESUMO
Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.
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Actinas , Recuperação de Fluorescência Após Fotodegradação , Recuperação de Fluorescência Após Fotodegradação/métodos , Actinas/análise , Animais , Polimerização , Neuritos , Neurônios/metabolismo , Neurônios/química , Células Cultivadas , Dendritos/química , Dendritos/metabolismoRESUMO
This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.
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Actinas , Junções Íntimas , Actinas/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Fosfoproteínas/metabolismoRESUMO
Botrytis cinerea is a necrotrophic fungus that can cause gray mold in over 1400 plant species. Once it is detected by Arabidopsis thaliana, several defense responses are activated against this fungus. The proper activation of these defenses determines plant susceptibility or resistance. It has been proposed that the RAC/ROP small GTPases might serve as a molecular link in this process. In this study, we investigate the potential role of the Arabidopsis RAC7 gene during infection with B. cinerea. For that, we evaluated A. thaliana RAC7-OX lines, characterized by the overexpression of the RAC7 gene. Our results reveal that these RAC7-OX lines displayed increased susceptibility to B. cinerea infection, with enhanced fungal colonization and earlier lesion development. Additionally, they exhibited heightened sensitivity to bacterial infections caused by Pseudomonas syringae and Pectobacterium brasiliense. By characterizing plant canonical defense mechanisms and performing transcriptomic profiling, we determined that RAC7-OX lines impaired the plant transcriptomic response before and during B. cinerea infection. Global pathway analysis of differentially expressed genes suggested that RAC7 influences pathogen perception, cell wall homeostasis, signal transduction, and biosynthesis and response to hormones and antimicrobial compounds through actin filament modulation. Herein, we pointed out, for first time, the negative role of RAC7 small GTPase during A. thaliana-B. cinerea interaction.
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Arabidopsis , Proteínas Monoméricas de Ligação ao GTP , Citoesqueleto de Actina , Arabidopsis/genética , Sistema Imunitário , Proteínas Monoméricas de Ligação ao GTP/genética , Transdução de SinaisRESUMO
AIM: We aimed to investigate the mechanisms involved in the neurotoxic effects of NDGA on differentiated and undifferentiated human neuroblastoma cells (MSN), assessing cell viability, changes in the actin cytoskeleton, cell migration and the expression of the 5-LOX enzyme and the inhibitor of cell cycle progression p21WAF1/CIP1. BACKGROUND: High expression and activity of the lipoxygenase enzyme (LOX) have been detected in several tumors, including neuroblastoma samples, suggesting the use of LOX inhibitors as potential therapy molecules. Among these, the natural compound nordihydroguaiaretic acid (NDGA) has been extensively tested as an antiproliferative drug against diverse types of cancer cells. OBJECTIVE: In this study, we analyzed the toxic effect of NDGA on neuroblastoma cells at a dose that did not affect cell survival when they differentiated to a neuron-like phenotype and the potential mechanisms involved in the anticancer properties. METHODS: We exposed human neuroblastoma cells (MSN) to different concentrations of NDGA before and after a differentiation protocol with retinoic acid and nerve growth factor and analyzed cell viability, cell migration, actin cytoskeleton morphology and the levels of the cell cycle inhibitor p21WAF1/CIP1 and 5-LOX. RESULTS: We found that differentiated human neuroblastoma cells are more resistant to NDGA than undifferentiated cells. The toxic effects of NDGA were accompanied by reduced cell migration, changes in actin cytoskeleton morphology, induction of p21WAF1/CIP1 and decreased levels of the 5-LOX enzyme. CONCLUSION: This study provides new evidence regarding the potential use of NDGA to induce cell death in human neuroblastoma.
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Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Masoprocol , Neuroblastoma , Humanos , Neuroblastoma/patologia , Masoprocol/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Relação Dose-Resposta a Droga , Tretinoína/farmacologia , Inibidores de Lipoxigenase/farmacologia , Antineoplásicos/farmacologiaRESUMO
Actin remodeling is a critical regulator of mast cell secretion. In previous work, we have shown that dehydroleucodine and xanthatin, two natural α,ß-unsaturated lactones, exhibit anti-inflammatory and mast cell stabilizing properties. Based on this background, this study aimed to determine whether the mast cell stabilizing action of these lactones is associated with changes in the actin cytoskeleton. Rat peritoneal mast cells were preincubated in the presence of dehydroleucodine or xanthatin before incubation with compound 48/80. Comparative studies with sodium cromoglycate and latrunculin B were also made. After treatments, different assays were performed on mast cell samples: ß-hexosaminidase release, cell viability studies, quantification of mast cells and their state of degranulation by light microscopy, transmission electron microscopy, and actin staining for microscopy observation. Results showed that dehydroleucodine and xanthatin inhibited mast cell degranulation, evidenced by the inhibition of ß-hexosaminidase release and decreased degranulated mast cell percentage. At the same time, both lactones altered the F-actin cytoskeleton in mast cells resulting, similarly to Latrunculin B, in a higher concentration of nuclear F-actin when activated by compound 48/80. For the first time, this study describes the biological properties of dehydroleucodine and xanthatin concerning to the rearrangement of actin filaments during stimulated exocytosis in mast cells. These data have important implications for developing new anti-inflammatory and mast cell stabilizing drugs and for designing new small molecules that may interact with the actin cytoskeleton.
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IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.
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Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown completely eliminated ROCKi's p53-dependent actions, since ROCK inhibition specifically elevated Rac-GTP levels only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor in improving the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.
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Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Dano ao DNA , Linhagem Celular TumoralRESUMO
BACKGROUND: As a progressive cerebrovascular disease, Moyamoya Disease (MMD) is a common cause of stroke in children and adults. However, the early biomarkers and pathogenesis of MMD remain poorly understood. METHODS AND MATERIAL: This study was conducted using plasma exosome samples from MMD patients. Next-generation high-throughput sequencing, real-time quantitative PCR, gene ontology analysis, and Kyoto Encyclopaedia of Genes and Genomes pathway analysis of ideal exosomal miRNAs that could be used as potential biomarkers of MMD were performed. The area under the Receiver Operating Characteristic (ROC) curve was used to evaluate the sensitivity and specificity of biomarkers for predicting events. RESULTS: Exosomes were successfully isolated and miRNA-sequence analysis yielded 1,002 differentially expressed miRNAs. Functional analysis revealed that they were mainly enriched in axon guidance, regulation of the actin cytoskeleton and the MAPK signaling pathway. Furthermore, 10 miRNAs (miR-1306-5p, miR-196b-5p, miR-19a-3p, miR-22-3p, miR-320b, miR-34a-5p, miR-485-3p, miR-489-3p, miR-501-3p, and miR-487-3p) were found to be associated with the most sensitive and specific pathways for MMD prediction. CONCLUSIONS: Several plasma secretory miRNAs closely related to the development of MMD have been identified, which can be used as biomarkers of MMD and contribute to differentiating MMD from non-MMD patients before digital subtraction angiography.
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MicroRNAs , Doença de Moyamoya , Adulto , Criança , Humanos , MicroRNAs/genética , Projetos Piloto , Doença de Moyamoya/diagnóstico , Doença de Moyamoya/genética , BiomarcadoresRESUMO
The non-cholinergic molecular targets of organophosphate (OP) compounds have recently been investigated to explain their role in the generation of non-neurological diseases, such as immunotoxicity and cancer. Here, we evaluated the effects of malathion and its dialkylphosphate (DAP) metabolites on the cytoskeleton components and organization of RAW264.7 murine macrophages as non-cholinergic targets of OP and DAPs toxicity. All OP compounds affected actin and tubulin polymerization. Malathion, dimethyldithiophosphate (DMDTP) dimethylthiophosphate (DMTP), and dimethylphosphate (DMP) induced elongated morphologies and the formation of pseudopods rich in microtubule structures, and increased filopodia formation and general actin disorganization in RAW264.7 cells and slightly reduced stress fibers in the human fibroblasts GM03440, without significantly disrupting the tubulin or vimentin cytoskeleton. Exposure to DMTP and DMP increased cell migration in the wound healing assay but did not affect phagocytosis, indicating a very specific modification in the organization of the cytoskeleton. The induction of actin cytoskeleton rearrangement and cell migration suggested the activation of cytoskeletal regulators such as small GTPases. We found that DMP slightly reduced Ras homolog family member A activity but increased the activities of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) from 5 min to 2 h of exposure. Chemical inhibition of Rac1 with NSC23766 reduced cell polarization and treatment with DMP enhanced cell migration, but Cdc42 inhibition by ML-141 completely inhibited the effects of DMP. These results suggest that methylated OP compounds, especially DMP, can modify macrophage cytoskeleton function and configuration via activation of Cdc42, which may represent a potential non-cholinergic molecular target for OP compounds.
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Inseticidas , Malation , Camundongos , Humanos , Animais , Malation/toxicidade , Malation/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Tubulina (Proteína)/metabolismo , Citoesqueleto de Actina/metabolismo , Inseticidas/toxicidade , Inseticidas/metabolismo , Movimento Celular , Compostos Organofosforados/metabolismo , Organofosfatos/metabolismoRESUMO
Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.
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Citrulinação , Armadilhas Extracelulares , Actinas/metabolismo , Ionomicina/farmacologia , Ionomicina/metabolismo , Proteínas Nucleares/metabolismo , Ligantes , Proteômica , Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Citoesqueleto/metabolismoRESUMO
Astrocytes are active participants in the performance of the Central Nervous System (CNS) in both health and disease. During aging, astrocytes are susceptible to reactive astrogliosis, a molecular state characterized by functional changes in response to pathological situations, and cellular senescence, characterized by loss of cell division, apoptosis resistance, and gain of proinflammatory functions. This results in two different states of astrocytes, which can produce proinflammatory phenotypes with harmful consequences in chronic conditions. Reactive astrocytes and senescent astrocytes share morpho-functional features that are dependent on the organization of the cytoskeleton. However, such changes in the cytoskeleton have yet to receive the necessary attention to explain their role in the alterations of astrocytes that are associated with aging and pathologies. In this review, we summarize all the available findings that connect changes in the cytoskeleton of the astrocytes with aging. In addition, we discuss future avenues that we believe will guide such a novel topic.
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Astrócitos , Citoesqueleto , Astrócitos/patologia , Microtúbulos , Sistema Nervoso Central/patologiaRESUMO
Highly focused near-infrared (NIR) lasers have been used to induce fibroblast and neuron protrusions in a technique called optical guidance. However, little is known about the biochemical and biophysical effects that the laser provokes in the cell and optimal protocols of stimulation have not yet been established. Using intermittent NIR laser radiation and multivariate time series representations of cell leading edge movement, we analyzed the direction and velocity of cell protrusions. We found that the orientation and advance of PC12 neuron phenotype cells and 3T3 fibroblasts protrusions remain after the laser is turned off, but the observed increase in velocity stops when radiation ceases. For an increase in the speed and distance of cell protrusions by NIR laser irradiation, the cell leading edge needs to be advancing prior to the stimulation, and NIR irradiation does not enable the cell to switch between retracting and advancing states. Using timelapse imaging of actin-GFP, we observed that NIR irradiation induces a faster recruitment of actin, promoting filament formation at the induced cell protrusions. These results provide fresh evidence to understand the phenomenon of the optical guidance of cell protrusions.
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Actinas , Luz , Fibroblastos , Citoesqueleto , LasersRESUMO
Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.
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Cavéolas , Traumatismo por Reperfusão , Ratos , Animais , Cavéolas/metabolismo , Actinas/metabolismo , Caveolina 3/metabolismo , Citoesqueleto/metabolismo , Caveolina 1/metabolismo , Traumatismo por Reperfusão/metabolismo , Mitocôndrias/metabolismoRESUMO
BACKGROUND: Previous studies have shown that the ability of tumor cells to move and migrate is related to the molecular chain pathway mediated by actin. This study focused on the molecular mechanism of gelsolin (GSN) as an important actin-binding protein in promoting HCC invasion and metastasis. METHODS: The relationship between GSN expression and clinical characteristics was observed by immunohistochemistry (IHC). In vitro and in vivo experiments confirmed the role of GSN in HCC metastasis. Dual-immunoprecipitation (IP), immunofluorescence (IF), western blotting, and the gelatinase activity assay were used to investigate the mechanism of GSN-promoting metastasis. PEX fusion proteins were used to intervene in the transfer molecular chain. RESULTS: Our study found that GSN promoted HCC invasion and metastasis through its synergistic effect with actin-related transfer molecular chain (actin-CD44-MMPs). Concretely, as an important binding molecule of actin, GSN activated MMP2 by interacting with MMP14. Furthermore, CD44 might be a key node in the above-mentioned mechanism. The use of MMP14 domain (PEX fusion protein) to competitively bind to CD44 helped to inhibit the activation of downstream MMP2. CONCLUSIONS: GSN played crucial roles in HCC metastatic process. An improved understanding of the multiple effects of GSN in HCC might facilitate a deeper appreciation of GSN as an important HCC regulator. The study identified GSN and its regulated transfer molecular chain as potential therapeutic targets for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Gelsolina/genética , Gelsolina/metabolismo , Actinas , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Linhagem Celular TumoralRESUMO
Our ability to learn and remember depends on the active formation, remodeling, and elimination of synapses. Thus, the development and growth of synapses as well as their weakening and elimination are essential for neuronal rewiring. The structural reorganization of synaptic complexes, changes in actin cytoskeleton and organelle dynamics, as well as modulation of gene expression, determine synaptic plasticity. It has been proposed that dysregulation of these key synaptic homeostatic processes underlies the synaptic dysfunction observed in many neurodegenerative diseases. Much is known about downstream signaling of activated N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoazolepropionate receptors; however, other signaling pathways can also contribute to synaptic plasticity and long-lasting changes in learning and memory. The non-receptor tyrosine kinase c-Abl (ABL1) is a key signal transducer of intra and extracellular signals, and it shuttles between the cytoplasm and the nucleus. This review focuses on c-Abl and its synaptic and neuronal functions. Here, we discuss the evidence showing that the activation of c-Abl can be detrimental to neurons, promoting the development of neurodegenerative diseases. Nevertheless, c-Abl activity seems to be in a pivotal balance between healthy synaptic plasticity, regulating dendritic spines remodeling and gene expression after cognitive training, and synaptic dysfunction and loss in neurodegenerative diseases. Thus, c-Abl genetic ablation not only improves learning and memory and modulates the brain genetic program of trained mice, but its absence provides dendritic spines resiliency against damage. Therefore, the present review has been designed to elucidate the common links between c-Abl regulation of structural changes that involve the actin cytoskeleton and organelles dynamics, and the transcriptional program activated during synaptic plasticity. By summarizing the recent discoveries on c-Abl functions, we aim to provide an overview of how its inhibition could be a potentially fruitful treatment to improve degenerative outcomes and delay memory loss.