RESUMO
Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.
Assuntos
Actinas , Contração Muscular , Cobaias , Animais , Contração Muscular/fisiologia , Actinas/metabolismo , Citocalasina D/farmacologia , Citocalasina D/metabolismo , Ceco/metabolismo , Artérias Carótidas/metabolismo , Cálcio/metabolismoRESUMO
Calorie restriction (CR) mimetic, resveratrol (RSV), has the capacity of promoting phagocytosis. However, its role in hepatic ischemia and reperfusion injury (HIRI) remains poorly understood. This study aimed to investigate the effect of RSV on alleviating HIRI and explore the underlying mechanisms. RSV was intraperitoneally injected in mice HIRI model, while RSV was co-incubated with culture medium for 24 h in RAW 264.7 cells and kupffer cells. Macrophage efferocytosis was assessed by immunostaining of PI and F4/80. The clearance of apoptotic neutrophils in the liver was determined by immunostaining of Ly6-G and cleaved-caspase-3. HE staining, Suzuki's score, serum levels of ALT, AST, TNF-α and IL-1ß were analyzed to evaluate HIRI. The efferocytosis inhibitor, Cytochalasin D, was utilized to investigate the effect of RSV on HIRI. Western blot was employed to measure the levels of AMPKα, phospho-AMPKα, STAT3, phospho-STAT3 and S1PR1. SiSTAT3 and inhibitors targeting AMPK, STAT3 and S1PR1, respectively, were used to confirm the involvement of AMPK/STAT3/S1PR1 pathway in RSV-mediated efferocytosis and HIRI. RSV facilitated the clearance of apoptotic neutrophils and attenuated HIRI, which was impeded by Cytochalasin D. RSV boosted macrophage efferocytosis by up-regulating the levels of phospho-AMPKα, phospho-STAT3 and S1PR1, which was reversed by AMPK, STAT3 and S1PR1 inhibitors, respectively. Inhibition of STAT3 suppressed RSV-induced clearance of apoptotic neutrophils and exacerbated HIRI. CR mimetic, RSV, alleviates HIRI by promoting macrophages efferocytosis through AMPK/STAT3/S1PR1 pathway, providing valuable insights into the mechanisms underlying the protective effects of CR on attenuating HIRI.
Assuntos
Proteínas Quinases Ativadas por AMP , Traumatismo por Reperfusão , Camundongos , Animais , Resveratrol/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Eferocitose , Restrição Calórica , Citocalasina D/metabolismo , Fígado/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/metabolismo , Macrófagos/metabolismo , IsquemiaRESUMO
BACKGROUND: Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study. METHODS: Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy. RESULT: Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels. CONCLUSION: Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.
Assuntos
Plaquetas , Células Endoteliais , Humanos , Apoptose , Plaquetas/metabolismo , Citocalasina D/farmacologia , Citocalasina D/metabolismo , Células Endoteliais/metabolismo , Regulação para CimaRESUMO
In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.
Assuntos
Actinas , Proteínas de Membrana , Masculino , Humanos , Actinas/metabolismo , Citocalasina D/farmacologia , Citocalasina D/análise , Citocalasina D/metabolismo , Proteínas de Membrana/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Imunoglobulinas/metabolismoRESUMO
BACKGROUND: It has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages. METHODS: Human THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). RESULTS: The cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data. CONCLUSIONS: Interventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.
Assuntos
Aterosclerose , Inflamassomos , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Citocalasina D/metabolismo , Citocalasina D/farmacologia , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/farmacologia , Lipoproteínas LDL/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos , Autofagia , Apoptose , Aterosclerose/genética , Aterosclerose/metabolismo , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , RNA/metabolismoRESUMO
Although it is widely recognized that disruption of ALS3 reduces the invasion of Candida albicans germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored. C. albicans strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and C. albicans cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a C. albicans strain that did not produce Als3 on its surface. Invasion via active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased C. albicans adhesion to cultured epithelial cell monolayers, presumably via increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in C. albicans invasion into mammalian oral epithelial cells.
Assuntos
Candida albicans , Proteínas Fúngicas , Animais , Candida albicans/genética , Citocalasina D/metabolismo , Citocalasina D/farmacologia , Células Epiteliais/microbiologia , Proteínas Fúngicas/metabolismo , Humanos , Mamíferos/metabolismo , Peptídeos/metabolismo , Timerosal/metabolismoRESUMO
Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.
Assuntos
Peptídeos Penetradores de Células , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Cátions/farmacologia , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Citocalasina D/metabolismo , Citocalasina D/farmacologia , Endocitose , Oligomicinas/farmacologia , TranscitoseRESUMO
Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.
Assuntos
Actinas , Mycobacterium tuberculosis , Humanos , Actinas/metabolismo , Citocalasina D/farmacologia , Citocalasina D/metabolismo , Citoesqueleto de Actina/metabolismo , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Colesterol/metabolismo , EsfingolipídeosRESUMO
Our previous study demonstrated mechanical stretch (MS) could induce the apoptosis of retinal pigment epithelial (RPE) cells, but the related mechanisms remained unclear. This study was to characterize the protein expression profile in RPE cell line ARPE-19 exposed to MS, cytochalasin D (CD; an inhibitor of actin polymerization) or CD + MS at 2-time points (6, 24 hr; n = 3, at each time point) by using proteomics technique. Our data highlighted that compared with control, ECE1 was continuously downregulated in ARPE-19 cells treated by MS or CD + MS from 6 to 24 hr. Function and protein-protein interaction network analyses showed ATAD2 was downregulated in all three treatment groups compared with control, but successive upregulation of RPS13 and RPL7 and downregulation of AHSG were specifically induced by MS. ATAD2 was enriched in cell cycle; AHSG was associated with membrane organization; RPS13 and RPL7 participated in ribosome biogenesis. Furthermore, transcription factor CREB1 that was upregulated in MS group at 24 hr after treatment, may negatively regulate ATAD2. The expressions of all crucial proteins in ARPE-19 cells were confirmed by western blot analysis. Overexpression of ATAD2 and AHSG were also shown to reverse the apoptosis of ARPE-19 cells induced by MS or CD + MS, with significantly decreased apoptotic rates and caspase-3 activities. Accordingly, our findings suggest downregulation of ATAD2 and AHSG may be potential contributors to the apoptosis of RPE cells induced by MS. Overexpression of them may represent underlying preventive and therapeutic strategies for MS-induced retinal disorders.
Assuntos
Apoptose/fisiologia , Células Epiteliais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocalasina D/metabolismo , Regulação para Baixo/fisiologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Mapas de Interação de Proteínas/fisiologia , Proteômica/métodos , Epitélio Pigmentado da Retina/fisiologia , Estresse Mecânico , Regulação para Cima/fisiologia , alfa-2-Glicoproteína-HS/metabolismoRESUMO
Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts. Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP. Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.
Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Processamento de Proteína Pós-Traducional , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amidas , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Citocalasina D/metabolismo , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Histonas/metabolismo , Humanos , Masculino , Piridinas , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tiazolidinas/metabolismoRESUMO
Traditionally, the screening of metabolites in microbial matrices is performed by monocultures. Nonetheless, the absence of biotic and abiotic interactions generally observed in nature still limit the chemical diversity and leads to "poorer" chemical profiles. Nowadays, several methods have been developed to determine the conditions under which cryptic genes are activated, in an attempt to induce these silenced biosynthetic pathways. Among those, the one strain, many compounds (OSMAC) strategy has been applied to enhance metabolic production by a systematic variation of growth parameters. The complexity of the chemical profiles from OSMAC experiments has required increasingly robust and accurate techniques. In this sense, deconvolution-based 1 HNMR quantification have emerged as a promising methodology to decrease complexity and provide a comprehensive perspective for metabolomics studies. Our present work shows an integrated strategy for the increased production and rapid quantification of compounds from microbial sources. Specifically, an OSMAC design of experiments (DoE) was used to optimize the microbial production of bioactive fusaric acid, cytochalasin D and 3-nitropropionic acid, and Global Spectral Deconvolution (GSD)-based 1 HNMR quantification was carried out for their measurement. The results showed that OSMAC increased the production of the metabolites by up to 33% and that GSD was able to extract accurate NMR integrals even in heavily coalescence spectral regions. Moreover, GSD-1 HNMR quantification was reproducible for all species and exhibited validated results that were more selective and accurate than comparative methods. Overall, this strategy up-regulated important metabolites using a reduced number of experiments and provided fast analyte monitor directly in raw extracts.
Assuntos
Técnicas de Cultura de Células/métodos , Citocalasina D/metabolismo , Ácido Fusárico/biossíntese , Metabolômica/métodos , Nitrocompostos/metabolismo , Propionatos/metabolismo , Ascomicetos/isolamento & purificação , Ascomicetos/metabolismo , Citocalasina D/análise , Ácido Fusárico/análise , Nitrocompostos/análise , Propionatos/análise , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The present study aimed to evaluate the effects of Krüppellike factor 2 (KLF2) on the differentiation of endothelial progenitor cells (EPCs) to endothelial cells (ECs) induced by shear stress, and to investigate the corresponding mechanisms. Cultured rat late EPCs were exposed to shear stress (12 dyn/cm2) for different lengths of time. Reverse transcriptionquantitative polymerase chain reaction (RTqPCR) was used to measure the initial KLF2 mRNA levels in each group. Subsequently, the EPCs were treated with antiintegrin ß1 or ß3 antibodies to block integrin ß1 and ß3, respectively, or cytochalasin D to destroy Factin, and the subsequent expression levels of KLF2 in EPCs were measured. Then, KLF2 small interfering RNAs (siRNAs) were transfected into EPCs, and RTqPCR was used to measure the mRNA expression level of KLF2. Additionally, flow cytometry was applied to evaluate the protein levels of cluster of differentiation 31 (CD31) and the von Willebrand factor (vWF), and the regulatory effects of KLF2 in the promoter region of vWF were determined via a luciferase assay. High shear stress upregulated KLF2 expression, while blocking integrin ß1/ß3 or destroying Factin resulted in a corresponding decrease in KLF2 expression. Downregulation of KLF2 expression by siKLF2 inhibited the differentiation of EPCs to ECs under shear stress conditions, while the expression of ECspecific markers decreased, including CD31 and vWF. Various lengths of the vWF promoter region induced vWF expression, and EPCs cotransfected with KLF2 significantly increased the vWF expression levels compared with the group treated with vWF alone (P<0.01). In conclusion, shear stress may upregulate KLF2 expression, which may be associated with the integrinactin cytoskeleton system. Most importantly, the shear stressinduced differentiation of EPCs may be mediated by KLF2.
Assuntos
Diferenciação Celular/genética , Células Progenitoras Endoteliais/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Estresse Mecânico , Actinas/genética , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Citocalasina D/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Progenitoras Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Integrina beta1/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Ratos , Fator de von Willebrand/genéticaRESUMO
Fundamental biological processes are carried out by curved epithelial sheets that enclose a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. Here we combine measurements of epithelial tension and shape with theoretical modelling to show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme strains in the tissue are accommodated by highly heterogeneous strains at a cellular level, in seeming contradiction to the measured tensional uniformity. This phenomenon is reminiscent of superelasticity, a behaviour that is generally attributed to microscopic material instabilities in metal alloys. We show that in epithelial cells this instability is triggered by a stretch-induced dilution of the actin cortex, and is rescued by the intermediate filament network. Our study reveals a type of mechanical behaviour-which we term active superelasticity-that enables epithelial sheets to sustain extreme stretching under constant tension.
Assuntos
Elasticidade , Células Epiteliais/citologia , Actinas/metabolismo , Ligas , Animais , Fenômenos Biomecânicos , Células CACO-2 , Forma Celular , Tamanho Celular , Citocalasina D/metabolismo , Cães , Células Epiteliais/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Células Madin Darby de Rim Canino , PressãoRESUMO
Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.
Assuntos
Candida albicans/metabolismo , Candidíase/microbiologia , Proteínas Fúngicas/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Actinas/análise , Actinas/metabolismo , Candida albicans/citologia , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Citocalasina D/análise , Citocalasina D/metabolismo , Ergosterol/metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP90/análise , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Morfogênese , Regulação para Cima , Proteínas ras/análise , Proteínas ras/genéticaRESUMO
Our previous study has shown that three-dimensional (3D) culture decreases mesenchymal stem cell (MSC) size, leading to enhanced trafficking ability and reduced lung vascular obstructions. However, the underlying mechanisms are unclear. In this study, we proposed that 3D culture reduces MSC size by increasing vesicle excretion. Scanning electron microscope showed that 3D culture markedly increased the amount of membrane-bound vesicles on the cell surface. In consistence, tunable resistive pulse sensing quantifying analysis of vesicles in the culture medium indicated that there were higher levels of vesicles in the 3D culture MSC medium. 3D culture significantly lowered the level of actin polymerization (F-actin), suggestive of lowering actin skeleton tension may facilitate vesicle excretion. Indeed, treatment of MSCs with Cytochalasin D or functional blockade of integrin ß1 caused increased vesicle secretion and decreased cell sizes. Thus, our results suggest that 3D culture reduces MSC size by increasing vesicle excretion which is likely mediated by lowering cytoskeleton tension. Stem Cells 2018;36:286-292.
Assuntos
Técnicas de Cultura de Células/métodos , Citoesqueleto de Actina/metabolismo , Animais , Tamanho Celular , Citocalasina D/metabolismo , Humanos , Integrina beta1/metabolismo , Transdução de SinaisRESUMO
Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories.
Assuntos
Membrana Celular/metabolismo , Microscopia Intravital/métodos , Profilinas/metabolismo , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Citocalasina D/metabolismo , Difusão/efeitos dos fármacos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Microscopia Intravital/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Inibidores da Síntese de Ácido Nucleico/farmacologia , Profilinas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/instrumentação , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Strategies for musculoskeletal tissue regeneration apply adult mesenchymal stem/stromal cells (MSCs) that can be sourced from bone marrow- and lipo-aspirates. Adipose tissue-derived MSCs are more easily harvested in the large quantities required for skeletal tissue-engineering approaches, but are generally considered to be less osteogenic than bone marrow MSCs. Therefore, we tested a new molecular strategy to improve their osteogenic lineage-differentiation potential using the fungal metabolite cytochalasin D (CytoD). We show that CytoD, which may function by redistributing the intracellular location of ß-actin (ACTB), is a potent osteogenic stimulant as reflected by significant increases in alkaline phosphatase activity, extracellular matrix mineralization, and osteoblast-related gene expression (e.g., RUNX2, ALPL, SPARC, and TGFB3). RNA sequencing analyses of MSCs revealed that acute CytoD treatment (24 hours) stimulates a broad program of osteogenic biomarkers and epigenetic regulators. CytoD decreases mRNA and protein levels of the Polycomb chromatin regulator Enhancer of Zeste Homolog 2 (EZH2), which controls heterochromatin formation by mediating trimethylation of histone 3 lysine 27 (H3K27me3). Reduced EZH2 expression decreases cellular H3K27me3 marks indicating a global reduction in heterochromatin. We conclude that CytoD is an effective osteogenic stimulant that mechanistically functions by blocking both cytoplasmic actin polymerization and gene-suppressive epigenetic mechanisms required for the acquisition of the osteogenic phenotype in adipose tissue-derived MSCs. This finding supports the use of CytoD in advancing the osteogenic potential of MSCs in skeletal regenerative strategies. Stem Cells Translational Medicine 2018;7:197-209.
Assuntos
Tecido Adiposo/citologia , Citocalasina D/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fungos/metabolismo , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Tecido Adiposo/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Epigênese Genética/fisiologia , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/fisiologia , Engenharia Tecidual/métodosRESUMO
Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.
Assuntos
Bacillus anthracis/fisiologia , Óptica e Fotônica/métodos , Esporos Bacterianos/metabolismo , Animais , Citocalasina D/metabolismo , Dispositivos Lab-On-A-Chip , Lasers , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Microscopia Confocal , Fagocitose , Células RAW 264.7 , Esporos Bacterianos/imunologia , Proteína Vermelha FluorescenteRESUMO
MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21-/- macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21-/- macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21-/- BMDMs in response to LPS or Listeria. TNF-α was increased in miR-21-/- BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21-/- cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21-/- macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21-/- BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21-/- mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.
Assuntos
Listeria monocytogenes/imunologia , Listeriose/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , MicroRNAs/metabolismo , Animais , Citocalasina D/metabolismo , Citocinas/metabolismo , Citoplasma/microbiologia , Expressão Gênica , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/imunologia , Substrato Quinase C Rico em Alanina Miristoilada , Óxido Nítrico/metabolismo , Fagocitose , Fator de Necrose Tumoral alfa/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The local mechanical properties of cells are frequently probed by force indentation experiments carried out with an atomic force microscope. Application of common contact models provides a single parameter, the Young's modulus, to describe the elastic properties of cells. The viscoelastic response of cells, however, is generally measured in separate microrheological experiments that provide complex shear moduli as a function of time or frequency. Here, we present a straightforward way to obtain rheological properties of cells from regular force distance curves collected in typical force indentation measurements. The method allows us to record the stress-strain relationship as well as changes in the weak power law of the viscoelastic moduli. We derive an analytical function based on the elastic-viscoelastic correspondence principle applied to Hertzian contact mechanics to model both indentation and retraction curves. Rheological properties are described by standard viscoelastic models and the paradigmatic weak power law found to interpret the viscoelastic properties of living cells best. We compare our method with atomic force microscopy-based active oscillatory microrheology and show that the method to determine the power law coefficient is robust against drift and largely independent of the indentation depth and indenter geometry. Cells were subject to Cytochalasin D treatment to provoke a drastic change in the power law coefficient and to demonstrate the feasibility of the approach to capture rheological changes extremely fast and precisely. The method is easily adaptable to different indenter geometries and acquires viscoelastic data with high spatiotemporal resolution.