RESUMO
Metabolic enzymes can adapt during energy stress, but the consequences of these adaptations remain understudied. Here, we discovered that hexokinase 1 (HK1), a key glycolytic enzyme, forms rings around mitochondria during energy stress. These HK1-rings constrict mitochondria at contact sites with the endoplasmic reticulum (ER) and mitochondrial dynamics protein (MiD51). HK1-rings prevent mitochondrial fission by displacing the dynamin-related protein 1 (Drp1) from mitochondrial fission factor (Mff) and mitochondrial fission 1 protein (Fis1). The disassembly of HK1-rings during energy restoration correlated with mitochondrial fission. Mechanistically, we identified that the lack of ATP and glucose-6-phosphate (G6P) promotes the formation of HK1-rings. Mutations that affect the formation of HK1-rings showed that HK1-rings rewire cellular metabolism toward increased TCA cycle activity. Our findings highlight that HK1 is an energy stress sensor that regulates the shape, connectivity, and metabolic activity of mitochondria. Thus, the formation of HK1-rings may affect mitochondrial function in energy-stress-related pathologies.
Assuntos
Dinaminas , Metabolismo Energético , Hexoquinase , Mitocôndrias , Dinâmica Mitocondrial , Proteínas Mitocondriais , Hexoquinase/metabolismo , Hexoquinase/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Dinaminas/metabolismo , Dinaminas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Animais , Trifosfato de Adenosina/metabolismo , Estresse Fisiológico , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Ciclo do Ácido Cítrico , Glucose-6-Fosfato/metabolismo , Camundongos , Células HeLa , Células HEK293 , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , MutaçãoRESUMO
The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.
Assuntos
Neoplasias da Mama , Proteínas Nucleares , RNA Longo não Codificante , Proteínas de Ligação a RNA , Fatores de Transcrição , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Feminino , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.
Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/químicaRESUMO
Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl-/-) and platelet-specific Mgl-deficient (platMgl-/-) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl3-induced injury was markedly reduced in Mgl-/- mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl-/- mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl-/- mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis.
Assuntos
Monoacilglicerol Lipases , Monoglicerídeos , Animais , Camundongos , Endocanabinoides/metabolismo , Lipólise , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoacilglicerol Lipases/genéticaRESUMO
Mitochondria actively contribute to cellular Ca2+ homeostasis. The molecular mechanisms of mitochondrial Ca2+ uptake and release are well characterized and are attributed to the multi-protein assembly of the mitochondrial Ca2+ uniporter complex (MCUC) and the mitochondrial sodium-calcium exchanger (NCLX), respectively. Hence, Ca2+ transfer from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE) into the mitochondrial matrix has been quantitatively visualized on the subcellular level using targeted fluorescent biosensors. However, a correlation between the amplitude of cytosolic Ca2+ elevation with that in the mitochondrial matrix has not been investigated in detail so far. In the present study, we combined the Ca2+-mobilizing agonist histamine with the H1-receptor antagonist risperidone to establish a well-tunable experimental approach allowing the correlation between low, slow, high, and fast cytosolic and mitochondrial Ca2+ signals in response to inositol 1,4,5-trisphosphate (IP3)-triggered ER Ca2+ release. Our present data confirm a defined threshold in cytosolic Ca2+, which is necessary for the activation of mitochondrial Ca2+ uptake. Moreover, our data support the hypothesis of different modes of mitochondrial Ca2+ uptake depending on the source of the ion (i.e., ER vs SOCE).
Assuntos
Sinalização do Cálcio , Risperidona , Risperidona/farmacologia , Risperidona/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismoRESUMO
p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory-Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62- total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-ß-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.
Assuntos
Queratinas/metabolismo , Agregados Proteicos , Proteína Sequestossoma-1/metabolismo , Animais , Células CHO , Cricetulus , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Sequestossoma-1/genéticaRESUMO
Ca2+-dependent gene regulation controls several functions to determine the fate of the cells. Proteins of the nuclear factor of activated T-cells (NFAT) family are Ca2+ sensitive transcription factors that control the cell growth, proliferation and insulin secretion in ß-cells. Translocation of NFAT proteins to the nucleus occurs in a sequence of events that starts with activating calmodulin-dependent phosphatase calcineurin in a Ca2+-dependent manner, which dephosphorylates the NFAT proteins and leads to their translocation to the nucleus. Here, we examined the role of IP3-generating agonists and near-UV light in the induction of NFATc3 migration to the nucleus in the pancreatic ß-cell line INS-1. Our results show that IP3 generation yields cytosolic Ca2+ rise and NFATc3 translocation. Moreover, near-UV light exposure generates reactive oxygen species (ROS), resulting in cytosolic Ca2+ spiking via the L-type Ca2+ channel and triggers NFATc3 translocation to the nucleus. Using the mitochondria as a Ca2+ buffering tool, we showed that ROS-induced cytosolic Ca2+ spiking, not the ROS themselves, was the triggering mechanism of nuclear import of NFATc3. Collectively, this study reveals the mechanism of near-UV light induced NFATc3 migration.
Assuntos
Sinalização do Cálcio , Fatores de Transcrição NFATC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta , Animais , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Inositol 1,4,5-Trifosfato/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos da radiação , RatosRESUMO
BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.
Assuntos
Retículo Endoplasmático/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Presenilina-1/metabolismo , Animais , Antracenos/farmacologia , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , MAP Quinase Quinase 4/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
BACKGROUND/AIMS: In pancreatic ß-cells, the intracellular Ca²âº homeostasis is an essential regulator of the cells major functions. The endoplasmic reticulum (ER) as interactive intracellular Ca²âº store balances cellular Ca²âº. In this study basal ER Ca²âº homeostasis was evaluated in order to reveal potential ß-cell-specificity of ER Ca²âº handling and its consequences for mitochondrial Ca²âº, ATP and respiration. METHODS: The two pancreatic cell lines INS-1 and MIN-6, freshly isolated pancreatic islets, and the two non-pancreatic cell lines HeLA and EA.hy926 were used. Cytosolic, ER and mitochondrial Ca²âº and ATP measurements were performed using single cell fluorescence microscopy and respective (genetically-encoded) sensors/dyes. Mitochondrial respiration was monitored by respirometry. GSK3ß activity was measured with ELISA. RESULTS: An atypical ER Ca²âº leak was observed exclusively in pancreatic islets and ß-cells. This continuous ER Ca²âº efflux is directed to mitochondria and increases basal respiration and organellar ATP levels, is established by GSK3ß-mediated phosphorylation of presenilin-1, and is prevented by either knockdown of presenilin-1 or an inhibition/knockdown of GSK3ß. Expression of a presenlin-1 mutant that mimics GSK3ß-mediated phosphorylation established a ß-cell-like ER Ca²âº leak in HeLa and EA.hy926 cells. The ER Ca²âº loss in ß-cells was compensated at steady state by Ca²âº entry that is linked to the activity of TRPC3. CONCLUSION: Pancreatic ß-cells establish a cell-specific ER Ca²âº leak that is under the control of GSK3ß and directed to mitochondria, thus, reflecting a cell-specific intracellular Ca²âº handling for basal mitochondrial activity.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Presenilina-1/metabolismo , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Glicogênio Sintase Quinase 3 beta/genética , Células HeLa , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Fosforilação , Presenilina-1/genética , RatosRESUMO
Mitochondria are multifunctional organelles that essentially contribute to cell signaling by sophisticated mechanisms of communications. Live cell imaging studies showed that mitochondria are dynamic and complex structures that form ramified networks by directed movements, fission, and fusion events. There is emerging evidence that the morphology of mitochondria determines cellular functions and vice versa. Several intracellular signaling pathways and messengers including Ca2+ dynamically influence the architecture of mitochondria. Because electron microscopy cannot be utilized for an assessment of dynamics of mitochondrial morphology in intact cells, most studies were performed using wide-field or laser confocal fluorescence microscopies that, due to limitations of their spatial resolution, do not allow investigating sub-mitochondrial structures. Accordingly, our understanding of the dynamics of substructures of mitochondria is quite limited. Here, we present a robust super-resolution method to quantify the dynamics of mitochondrial cristae, the main substructures of the inner mitochondrial membrane, exploiting structured illumination microscopy (SIM). We observed that knockdown of the dynamin-like 120-kDa protein, which is encoded by the OPA1 gene, specifically reduces the dynamics of the mitochondrial cristae membranes (CM), while the inner boundary membrane (IBM) remained flexible. We further used dual color SIM to quantify the dynamics of CM in the junction between mitochondria and the endoplasmic reticulum (ER; mitochondrial associated membranes, MAMs). Intracellular Ca2+ release spatially reduced CM-dynamics in MAMs. Moreover, CM-dynamics was independent from matrix Ca2+ signal. Our data suggest that local Ca2+ signals specifically control CM-dynamics and structure to facilitate a well-balanced functional (Ca2+) interplay between mitochondria and the ER.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/fisiologia , Células HeLa , Humanos , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/fisiologiaRESUMO
Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.
Assuntos
Tecido Adiposo Marrom/metabolismo , Ácidos Graxos/metabolismo , Esterol Esterase/metabolismo , Termogênese , Acetilcoenzima A/metabolismo , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/ultraestrutura , Animais , Autofagia , Temperatura Corporal , Carnitina/análogos & derivados , Carnitina/metabolismo , Temperatura Baixa , Progressão da Doença , Dislipidemias/metabolismo , Dislipidemias/patologia , Metabolismo Energético , Glucose/metabolismo , Hipotermia Induzida , Gotículas Lipídicas/metabolismo , Lipólise , Masculino , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Oxirredução , Consumo de Oxigênio , Esterol Esterase/deficiência , Proteína Desacopladora 1/metabolismoRESUMO
During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.
Assuntos
Autofagia/fisiologia , Lipólise/fisiologia , Macrófagos Peritoneais/metabolismo , Triglicerídeos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Catepsina B/biossíntese , Catepsina B/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Lipase/genética , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/genética , Macrolídeos/farmacologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Mutantes , Esterol Esterase/genética , Esterol Esterase/metabolismo , Triglicerídeos/genéticaRESUMO
BACKGROUND/AIMS: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. METHODS: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. RESULTS: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. CONCLUSIONS: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Cálcio/agonistas , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estilbenos/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Transporte de Íons/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Especificidade de Órgãos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Resveratrol , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.
Assuntos
Cálcio/análise , Cálcio/química , Retículo Endoplasmático/química , Corantes Fluorescentes/química , Proteínas Luminescentes/química , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia ConfocalRESUMO
The precision of single-molecule localization-based super-resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV-induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single-color dSTORM. By screening 39â dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error-free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15â nm was achieved. Caged SD-dSTORM can resolve the ultrastructure of single 40â nm synaptic vesicles in brain sections similar to images obtained by immuno-electron microscopy, yet with much improved label density in two independent channels.
Assuntos
Encéfalo/ultraestrutura , Cor , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Vesículas Sinápticas/ultraestrutura , Animais , Encéfalo/citologia , Camundongos , Estrutura Molecular , Células NIH 3T3RESUMO
The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca2+ elevation hyperpolarizes the CM most likely caused by Ca2+ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.
Assuntos
Trifosfato de Adenosina , Potencial da Membrana Mitocondrial , Membranas Mitocondriais , Potencial da Membrana Mitocondrial/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Membranas Mitocondriais/metabolismo , Transdução de Sinais , Fosforilação Oxidativa , Cálcio/metabolismo , Mitocôndrias/metabolismo , Microscopia/métodos , HumanosRESUMO
In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.
RESUMO
We established a breast cancer cell line from a fast growing mouse WAP-SVT/t breast tumor. Cells from this line, SVTneg2, switched off T-antigen expression, carry a missense mutation at the p53 codon 242 (mouse G242 corresponds to human hot spot mutation G245), are malignantly transformed, highly aneuploid and very insensitive to apoptotic stimuli. To examine the influence of wild-type p53 (wtp53) restoration on the behavior of the SVTneg2 cells, we transfected these cells with wtp53 and generated three permanent cell lines expressing wtp53. Interestingly, restoration of p53 had no influence on chemotherapy sensitivity and the transformation capacity of these breast cancer cells, but markedly changed the gene expression of wtp53-dependent genes after doxorubicin treatment. We postulate that restoration of p53 leads to massive changes in gene expression and to a reduced proliferation rate, but is not sufficient to overcome the malignant phenotype and the chemoresistance of SVTneg2.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica , Neoplasias Mamárias Experimentais/metabolismo , Proteína Supressora de Tumor p53/genética , Aneuploidia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Sequência Consenso , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Fenótipo , Ligação Proteica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The mitochondrial inner boundary membrane harbors a protein called MICU1, which is sensitive to Ca2+ and binds to the MICOS components Mic60 and CHCHD2. Changes in the mitochondrial cristae junction structure and organization in MICU1-/- cells lead to increased cytochrome c release, membrane potential rearrangement, and changes in mitochondrial Ca2+ uptake dynamics. These findings shed new light on the multifaceted role of MICU1, highlighting its involvement not only as an interaction partner and regulator of the MCU complex but also as a crucial determinant of mitochondrial ultrastructure and, thus, an essential player in processes initiating apoptosis.
Assuntos
Citocromos c , Proteínas de Transporte da Membrana Mitocondrial , Citocromos c/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
Mitochondrial uncoupling proteins UCP1 and UCP2 have a structural homology of app. 60%. They execute their mitochondria uncoupling function through different molecular mechanisms. Non-shivering thermogenesis by UCP1 is mediated through a transmembrane dissipation of the proton motive force to create heat during sympathetic stimulation. UCP2, on the other hand, modulates through the interaction with methylated MICU1 the permeability of the cristae junction, which acts as an isolator for the cristae-located mitochondrial membrane potential. In this mini-review, we discuss and compare the recently described molecular mechanism of UCP1 in brown adipose tissue and UCP2 in aged and cancer non-excitable cells that contribute to mitochondrial uncoupling, and the synergistic effects of both UCPs with the mitochondrial Ca2+ uptake machinery.