RESUMO
Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins-hydrophobins (HFBs)-that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.
Assuntos
Parede Celular/genética , Proteínas Fúngicas/genética , Esporos Fúngicos/genética , Trichoderma/genética , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Interações Hidrofóbicas e Hidrofílicas , Hifas/genética , Hifas/crescimento & desenvolvimento , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Esporos Fúngicos/crescimento & desenvolvimento , Trichoderma/crescimento & desenvolvimentoRESUMO
Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.
Assuntos
Resinas Epóxi/química , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica/métodos , Organelas/ultraestrutura , Tetróxido de Ósmio/química , Manejo de Espécimes/métodos , Animais , Células CHO , Cricetulus , Fluorescência , Imunofluorescência/métodos , Humanos , Microscopia de Fluorescência , Imagem Molecular , Organelas/metabolismoRESUMO
Pancreatic beta cells are insulin-producing cells that are structurally and functionally polarized in the islets of Langerhans. The organization and position of the Golgi complex play a key role in maintaining a polarized cell state, but the factors and molecular mechanisms determining the Golgi polarization of pancreatic beta cells are still unknown. In the current study, using pancreatic beta cell-specific Atg5 knockout mice, we found that Atg5, an essential gene for autophagy, plays a pivotal role in regulating Golgi integrity and polarization by affecting the expression of genes involved in vesicle transport. Deletion of Atg5 led to endoplasmic reticulum (ER) stress and impaired the distribution of proinsulin and insulin secretion of pancreatic beta cells, which further exacerbates diabetes. These results contribute to a comprehensive understanding of autophagy-mediated Golgi polarization and its regulation of the function of pancreatic beta cells.
Assuntos
Células Secretoras de Insulina , Animais , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Complexo de Golgi/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proinsulina/metabolismoRESUMO
OBJECTIVES: Sepsis remains a highly lethal disease, whereas the precise reasons for death remain poorly understood. Prokineticin2 is a secreted protein that regulates diverse biological processes. Whether prokineticin2 is beneficial or deleterious to sepsis and the underlying mechanisms remain unknown. DESIGN: Prospective randomized animal investigation and in vitro studies. SETTING: Research laboratory at a medical university hospital. SUBJECTS: Prokineticin2 deficiency and wild-type C57BL/6 mice were used for in vivo studies; sepsis patients by Sepsis-3 definitions, patient controls, and healthy controls were used to obtain blood for in vitro studies. INTERVENTIONS: Prokineticin2 concentrations were measured and analyzed in human septic patients, patient controls, and healthy individuals. The effects of prokineticin2 on sepsis-related survival, bacterial burden, organ injury, and inflammation were assessed in an animal model of cecal ligation and puncture-induced polymicrobial sepsis. In vitro cell models were also used to study the role of prokineticin2 on antibacterial response of macrophages. MEASUREMENTS AND MAIN RESULTS: Prokineticin2 concentration is dramatically decreased in the patients with sepsis and septic shock compared with those of patient controls and healthy controls. Furthermore, the prokineticin2 concentration in these patients died of sepsis or septic shock is significantly lower than those survival patients with sepsis or septic shock, indicating the potential value of prokineticin2 in the diagnosis of sepsis and septic shock, as well as the potential value in predicting mortality in adult patients with sepsis and septic shock. In animal model, recombinant prokineticin2 administration protected against sepsis-related deaths in both heterozygous prokineticin2 deficient mice and wild-type mice and alleviated sepsis-induced multiple organ damage. In in vitro cell models, prokineticin2 enhanced the phagocytic and bactericidal functions of macrophage through signal transducers and activators of transcription 3 pathway which could be abolished by signal transducers and activators of transcription 3 inhibitors S3I-201. Depletion of macrophages reversed prokineticin2-mediated protection against polymicrobial sepsis. CONCLUSIONS: This study elucidated a previously unrecognized role of prokineticin2 in clinical diagnosis and treatment of sepsis. The proof-of-concept study determined a central role of prokineticin2 in alleviating sepsis-induced death by regulation of macrophage function, which presents a new strategy for sepsis immunotherapy.
Assuntos
Sepse , Choque Séptico , Animais , Modelos Animais de Doenças , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Estudos ProspectivosRESUMO
Etoposide-induced protein 2.4 (EI24), located on the endoplasmic reticulum (ER) membrane, has been proposed to be an essential autophagy protein. Specific ablation of EI24 in neuronal and liver tissues causes deficiency of autophagy flux. However, the molecular mechanism of the EI24-mediated autophagy process is still poorly understood. Like neurons and hepatic cells, pancreatic ß cells are also secretory cells. Pancreatic ß cells contain large amounts of ER and continuously synthesize and secrete insulin to maintain blood glucose homeostasis. Yet, the effect of EI24 on autophagy of pancreatic ß cells has not been reported. Here, we show that the autophagy process is inhibited in EI24-deficient primary pancreatic ß cells. Further mechanistic studies demonstrate that EI24 is enriched at the ER-mitochondria interface and that the C-terminal domain of EI24 is important for the integrity of the mitochondria-associated membrane (MAM) and autophagy flux. Overexpression of EI24, but not the EI24-ΔC mutant, can rescue MAM integrity and decrease the aggregation of p62 and LC3II in the EI24-deficient group. By mass spectrometry-based proteomics following immunoprecipitation, EI24 was found to interact with voltage-dependent anion channel 1 (VDAC1), inositol 1,4,5-trisphosphate receptor (IP3R), and the outer mitochondrial membrane chaperone GRP75. Knockout of EI24 impairs the interaction of IP3R with VDAC1, indicating that these proteins may form a quaternary complex to regulate MAM integrity and the autophagy process.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Sistemas CRISPR-Cas , Células Cultivadas , Feminino , Células HEK293 , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Proteínas Nucleares/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
In the published article, few errors were noticed and this has been corrected with this erratum publication.
RESUMO
Low temporal resolution and limited photocontrollable fluorescent protein probes have restricted the widespread application of single-molecule localization microscopy (SMLM). In the current study, we developed a new photoconvertible fluorescent protein (PCFP), pcStar, and quick single molecule-guided Bayesian localization microscopy (Quick-SIMBA). The combination of pcStar and Quick-SIMBA achieved the highest temporal resolution (0.1-0.25 s) with large field-of-view (76 × 9.4 µm2 -76 × 31.4 µm2) among the SMLM methods, which enabled the dynamic movements of the endoplasmic reticulum dense tubular matrix to be resolved. Moreover, pcStar extended the application of SMLM to imaging the immediate early nanostructures in Drosophila embryos and revealed a specific "parallel three-pillar" structure in the neuronal-glial cell junction, helping to elucidate glial cell "locking" and support of neurons during Drosophila embryogenesis.
Assuntos
Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Imagem Individual de Molécula/métodos , Actinas/análise , Animais , Teorema de Bayes , Linhagem Celular , Drosophila/embriologia , Retículo Endoplasmático/ultraestrutura , Humanos , Microscopia de Fluorescência/métodosRESUMO
The molecular mechanism in pancreatic ß cells underlying hyperlipidemia and insulin insufficiency remains unclear. Here, we find that the fatty acid-induced decrease in insulin levels occurs due to a decrease in insulin translation. Since regulation at the translational level is generally mediated through RNA-binding proteins, using RNA antisense purification coupled with mass spectrometry, we identify a novel insulin mRNA-binding protein, namely, DDX1, that is sensitive to palmitate treatment. Notably, the knockdown or overexpression of DDX1 affects insulin translation, and the knockdown of DDX1 eliminates the palmitate-induced repression of insulin translation. Molecular mechanism studies show that palmitate treatment causes DDX1 phosphorylation at S295 and dissociates DDX1 from insulin mRNA, thereby leading to the suppression of insulin translation. In addition, DDX1 may interact with the translation initiation factors eIF3A and eIF4B to regulate translation. In high-fat diet mice, the inhibition of insulin translation happens at an early prediabetic stage before the elevation of glucose levels. We speculate that the DDX1-mediated repression of insulin translation worsens the situation of insulin resistance and contributes to the elevation of blood glucose levels in obese animals.
Assuntos
RNA Helicases DEAD-box/metabolismo , Ácidos Graxos/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Ácidos Graxos não Esterificados/metabolismo , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Oligonucleotídeos Antissenso/genética , Palmitatos/metabolismo , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Transdução de SinaisRESUMO
Superresolution microscopy (SM) techniques are among the revolutionary methods for molecular and cellular observations in the 21st century. SM techniques overcome optical limitations, and several new observations using SM lead us to expect these techniques to have a large impact on neuroscience in the near future. Several types of SM have been developed, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), each with special features. In this Minisymposium, experts in these different types of SM discuss the new structural and functional information about specific important molecules in neuroscience that has been gained with SM. Using these techniques, we have revealed novel mechanisms of endocytosis in nerve growth, fusion pore dynamics, and described quantitative new properties of excitatory and inhibitory synapses. Additional powerful techniques, including single molecule-guided Bayesian localization SM (SIMBA) and expansion microscopy (ExM), alone or combined with super-resolution observation, are also introduced in this session.
Assuntos
Encéfalo/citologia , Microscopia Eletrônica de Transmissão/métodos , Rede Nervosa/citologia , Neurociências/métodos , Imagem Óptica/métodos , Animais , Encéfalo/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão/tendências , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/tendências , Rede Nervosa/ultraestrutura , Neurociências/tendências , Imagem Óptica/tendênciasRESUMO
Calcium homeostasis is essential for maintaining the viability and function of pancreatic ß cells and plays a key role in preventing the development of diabetes. Decreased levels of ATPase sarcoplasmic/endoplasmic reticulum Ca2+-transporting 2 (ATP2a2), the main calcium pump in ß cells, are often found in individuals with diabetes and in diabetic animal models. However, the regulators of ATP2a2 and the molecular mechanisms responsible for controlling ATP2a2 activity remain unclear. Etoposide-induced protein 2.4 (Ei24) is also down-regulated in ß cells of diabetic individuals, whereas the effect of decreased Ei24 level on ß-cell function is not clarified. Here, using Cre-LoxP and CRISPR/Cas9-based genomic knockout (KO) approaches to generate pancreatic ß cell-specific Ei24 KO mice and pancreatic ß-cell lines, we found that Ei24 regulates ATP2a2 activity. Specifically, we observed that Ei24 binds to ATP2a2 through Ei24 residues 293-299, which we named here the ATP2a2-interacting region (AIR). Loss of Ei24 inactivated ATP2a2, disrupted calcium homeostasis, and deactivated the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-AMP-activated protein kinase (AMPK) pathway. Elevation of calcium concentration in the endoplasmic reticulum or agonist-induced AMPK activation rescued pancreatic ß-cell survival and improved glucose tolerance of Ei24 KO mice. Our findings indicate that targeting the Ei24-ATP2a2 interaction to increase ATP2a2 activity can protect pancreatic ß cells and improve glucose homeostasis in diabetic models, suggesting that Ei24 could potentially serve as a target to prevent or manage diabetes.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Células Secretoras de Insulina/citologia , Proteínas Nucleares/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Cálcio/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Sobrevivência Celular , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Metabolismo Energético , Técnicas de Inativação de Genes , Homeostase , Humanos , Células Secretoras de Insulina/patologia , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Ratos , Transdução de SinaisRESUMO
Motivation: Super-resolution fluorescence microscopy with a resolution beyond the diffraction limit of light, has become an indispensable tool to directly visualize biological structures in living cells at a nanometer-scale resolution. Despite advances in high-density super-resolution fluorescent techniques, existing methods still have bottlenecks, including extremely long execution time, artificial thinning and thickening of structures, and lack of ability to capture latent structures. Results: Here, we propose a novel deep learning guided Bayesian inference (DLBI) approach, for the time-series analysis of high-density fluorescent images. Our method combines the strength of deep learning and statistical inference, where deep learning captures the underlying distribution of the fluorophores that are consistent with the observed time-series fluorescent images by exploring local features and correlation along time-axis, and statistical inference further refines the ultrastructure extracted by deep learning and endues physical meaning to the final image. In particular, our method contains three main components. The first one is a simulator that takes a high-resolution image as the input, and simulates time-series low-resolution fluorescent images based on experimentally calibrated parameters, which provides supervised training data to the deep learning model. The second one is a multi-scale deep learning module to capture both spatial information in each input low-resolution image as well as temporal information among the time-series images. And the third one is a Bayesian inference module that takes the image from the deep learning module as the initial localization of fluorophores and removes artifacts by statistical inference. Comprehensive experimental results on both real and simulated datasets demonstrate that our method provides more accurate and realistic local patch and large-field reconstruction than the state-of-the-art method, the 3B analysis, while our method is more than two orders of magnitude faster. Availability and implementation: The main program is available at https://github.com/lykaust15/DLBI. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Aprendizado Profundo , Microscopia de Fluorescência , Software , Teorema de Bayes , Células/ultraestrutura , Simulação por ComputadorRESUMO
Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (â¼100 W/cm(2)) live-cell SR imaging at â¼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.
Assuntos
Rastreamento de Células/métodos , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Microscopia de Fluorescência/métodos , Luz , Razão Sinal-RuídoRESUMO
Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Proteínas Luminescentes/metabolismo , Microtúbulos/metabolismo , Nanotecnologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Sintaxina 1/metabolismoRESUMO
Pancreatic adenocarcinoma upregulated factor (PAUF) is a new oncogene that activates signaling pathways that play a critical role in resistance to gemcitabine. We thus speculated that PAUF also plays a role in resistance to gemcitabine of pancreatic cancer cells. We established BxPC-3 cell lines with stable PAUF knockdown (BxPC-3_shPAUF) and controls (BxPC-3_shCtrl) and evaluated sensitivity to gemcitabine in vitro by MTT and flow cytometry. We established a xenograft model of human pancreatic cancer to examine PAUF function in gemcitabine resistance in vivo. Gene chip microarrays were performed to identify differentially expressed genes in BxPC-3_shPAUF and BxPC-3_shCtrl cells. Silencing PAUF increased the sensitivity of BxPC-3 cells to gemcitabine in vitro and in vivo. PAUF-knockdown BxPC-3 cell lines treated with gemcitabine showed increased proliferation inhibition and apoptosis compared with controls. Gemcitabine exhibited a more pronounced effect on reduction of BxPC-3_shPAUF tumors than BxPC-3_shCtrl tumors. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays confirmed a significantly higher apoptotic rate of BXPC-3_shPAUF tumors compared with BXPC-3_shCtrl tumors. Gene array showed that PAUF function in gemcitabine sensitivity might involve MRP2, MRP3, MDR1, PIK3R1, and NFkB2 genes. PAUF could be considered as a key molecular target for sensitizing pancreatic cancer cells to gemcitabine.
Assuntos
Adenocarcinoma/patologia , Desoxicitidina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Lectinas/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular , Lectinas/genética , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.
Assuntos
Proteínas de Fluorescência Verde , Proteínas Luminescentes , Microscopia de Fluorescência/métodos , Engenharia de Proteínas/métodos , Animais , Células COS , Chlorocebus aethiops , Cromatografia em Gel , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Modelos Moleculares , Processos Fotoquímicos , Plasmídeos , Conformação Proteica , Transfecção , Proteína Vermelha FluorescenteRESUMO
Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.
Assuntos
Microscopia de Fluorescência/métodos , Linhagem Celular , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Mutação , Fotoquímica/métodos , Fótons , Espectrofotometria/métodosRESUMO
Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.
Assuntos
Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Imagem Molecular/métodos , Coloração e Rotulagem/métodos , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Via Secretória , Proteína Vermelha FluorescenteRESUMO
Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.
Assuntos
Biomarcadores/análise , Butiril-CoA Desidrogenase/análise , Proteínas de Caenorhabditis elegans/análise , Caenorhabditis elegans/metabolismo , Vesículas Citoplasmáticas/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Western Blotting , Vesículas Citoplasmáticas/ultraestrutura , Metabolismo dos Lipídeos , Lipídeos/química , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²âº entry via CRAC (Ca²âº- release-activated Ca²âº) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹6, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹6 residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans , Proteínas de Membrana/química , Tirosina/química , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Proteína ORAI1 , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Análise de Célula Única , Molécula 1 de Interação Estromal , Tirosina/genéticaRESUMO
Hydrogen peroxide (H2O2) functions as a signaling molecule in diverse cellular processes. While cells have evolved the capability to detect and manage changes in H2O2 levels, the mechanisms regulating key H2O2-producing enzymes to maintain optimal levels, especially in pancreatic beta cells with notably weak antioxidative defense, remain unclear. We found that the protein EI24 responds to changes in H2O2 concentration and regulates the production of H2O2 by controlling the translation of NOX4, an enzyme that is constitutively active, achieved by recruiting an RNA-binding protein, RTRAF, to the 3'-UTR of Nox4. Depleting EI24 results in RTRAF relocating into the nucleus, releasing the brake on NOX4 translation. The excessive production of H2O2 by liberated NOX4 further suppresses the translation of the key transcription factor MafA, ultimately preventing its binding to the Ins2 gene promoter and subsequent transcription of insulin. Treatment with a specific NOX4 inhibitor or the antioxidant NAC reversed these effects and alleviated the diabetic symptoms in beta-cell specific Ei24-KO mice. This study revealed a new mechanism through which cells regulate oxidative stress at the translational level, involving an ER-tethered RNA-binding protein that controls the expression of the key H2O2-producing enzyme NOX4.