RESUMEN
When cells with excess DNA, such as tetraploid cells, undergo cell division, it can contribute to cellular transformation via asymmetrical chromosome segregation-generated genetic diversity. Cell cycle progression of tetraploid cells is suppressed by large tumor suppressor 2 (LATS2) kinase-induced inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP). We recently reported that the oncogene v-Src induces tetraploidy and promotes cell cycle progression of tetraploid cells by suppressing LATS2 activity. We explore here the mechanism by which v-Src suppresses LATS2 activity and the role of LATS2 in v-Src-expressing cells. LATS2 was directly phosphorylated by v-Src and the proto-oncogene c-Src, resulting in decreased LATS2 kinase activity. This kinase-deficient LATS2 accumulated in a YAP transcriptional activity-dependent manner, and knockdown of either LATS2 or the LATS2-binding partner moesin-ezrin-radixin-like protein (Merlin) accelerated v-Src-induced membrane bleb formation. Upon v-Src expression, the interaction of Merlin with LATS2 was increased possibly due to a decrease in Merlin phosphorylation at Ser518, the dephosphorylation of which is required for the open conformation of Merlin and interaction with LATS2. LATS2 was colocalized with Merlin at the plasma membrane in a manner that depends on the Merlin-binding region of LATS2. The bleb formation in v-Src-expressing and LATS2-knockdown cells was rescued by the reexpression of wild-type or kinase-dead LATS2 but not the LATS2 mutant lacking the Merlin-binding region. These results suggest that the kinase-deficient LATS2 plays a role with Merlin at the plasma membrane in the maintenance of cortical rigidity in v-Src-expressing cells, which may cause tumor suppression.
Asunto(s)
Estructuras de la Membrana Celular/enzimología , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Estructuras de la Membrana Celular/genética , Células HCT116 , Células HT29 , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Proteína Oncogénica pp60(v-src)/genética , Proteínas Serina-Treonina Quinasas/genética , Proto-Oncogenes Mas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Señalizadoras YAPRESUMEN
A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.
Asunto(s)
Estructuras de la Membrana Celular/ultraestructura , Rhizobiaceae/clasificación , Rhizobiaceae/citología , Rhizobiaceae/aislamiento & purificación , Bacterias Anaerobias , Composición de Base , Estructuras de la Membrana Celular/genética , ADN Bacteriano/genética , Ácidos Grasos , Fermentación , Genoma Bacteriano , Sedimentos Geológicos/microbiología , Japón , Filogenia , ARN Ribosómico 16S , Rhizobiaceae/genética , Análisis de Secuencia de ADNRESUMEN
A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication.IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Hepacivirus/fisiología , Proteínas de la Membrana/metabolismo , ARN Viral/biosíntesis , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Línea Celular Tumoral , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/virología , Genotipo , Humanos , Proteínas de la Membrana/genética , ARN Viral/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Cell biology considers most animal tissues as assemblies of "individual" cells that rely on different contact-dependent communication mechanisms, including synapses, gap junctions or - a recent awareness - membrane nano- and microtubes. However, by protease-mediated singularization of dense 2D/ 3D cell cultures and tissue explants, we show here that cell collectives stay connected via a continuous meshwork of F-actin-based membrane tubes, resembling tunneling nanotube (TNT)-based networks observed between dispersed cell cultures. Fusion of respective tubes was accompanied by the ingrowth of microtubules and the invasion of mitochondria and lysosomes. Remarkably, in homology to the plasmodesmata-based plant symplast, we found evidence for expanded, membrane-based syncytia in animal tissues by observing dye transfer among the highly interlinked cells. This approach allows for the first time to visualize and quantify membrane continuity-based connections among densely packed cells and to assess their potential physiological and pathological impact closer to the in vivo situation.
Asunto(s)
Citoesqueleto de Actina/genética , Comunicación Celular/genética , Células Gigantes/fisiología , Péptido Hidrolasas/química , Citoesqueleto de Actina/fisiología , Actinas/genética , Actinas/fisiología , Animales , Comunicación Celular/fisiología , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/fisiología , Fibroblastos/fisiología , Uniones Comunicantes/genética , Uniones Comunicantes/fisiología , Morfogénesis/genética , Morfogénesis/fisiología , Nanotubos/química , Ratas , Sinapsis/genética , Sinapsis/fisiologíaRESUMEN
Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.
Asunto(s)
Actinas/metabolismo , Estructuras de la Membrana Celular/metabolismo , Macrófagos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Actinas/genética , Animales , Sistemas CRISPR-Cas , Estructuras de la Membrana Celular/genética , Eliminación de Gen , Lipopolisacáridos/farmacología , Ratones , Células RAW 264.7 , Proteínas de Unión al GTP rab/genéticaRESUMEN
Membrane blebbing-dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D-environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D-collagen, while RhoB overexpression enhanced blebbing and 3D-collagen migration in a manner dependent on its plasma membrane localization and down-stream effectors ROCK and Myosin II RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11-positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D-migration of ALL cells. In conclusion, KIF13A-mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Movimiento Celular , Cinesinas/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Línea Celular Tumoral , Estructuras de la Membrana Celular/genética , Colágeno/genética , Colágeno/metabolismo , Endosomas/genética , Endosomas/metabolismo , Humanos , Cinesinas/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteína de Unión al GTP rhoB/genéticaRESUMEN
The formation of neurites is an important process affecting the cognitive abilities of an organism. Neurite growth requires the addition of new membranes, but the metabolic remodeling necessary to supply lipids for membrane expansion is poorly understood. Here, we show that synaptic activity, one of the most important inducers of neurite growth, transcriptionally regulates the expression of neuronal glucose transporter Glut3 and rate-limiting enzymes of glycolysis, resulting in enhanced glucose uptake and metabolism that is partly used for lipid synthesis. Mechanistically, CREB regulates the expression of Glut3 and Siah2, the latter and LDH activity promoting the normoxic stabilization of HIF-1α that regulates the expression of rate-limiting genes of glycolysis. The expression of dominant-negative HIF-1α or Glut3 knockdown blocks activity-dependent neurite growth in vitro while pharmacological inhibition of the glycolysis and specific ablation of HIF-1α in early postnatal mice impairs the neurite architecture. These results suggest that the manipulation of neuronal glucose metabolism could be used to treat some brain developmental disorders.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Neuritas/metabolismo , Sinapsis/metabolismo , Animales , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/patología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Transportador de Glucosa de Tipo 3/biosíntesis , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Glucólisis/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Neuritas/patología , Ratas , Ratas Sprague-Dawley , Sinapsis/genética , Sinapsis/patología , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67â nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Polaridad Celular/fisiología , Células Epiteliales/metabolismo , Proteínas Ligadas a GPI/metabolismo , Modelos Biológicos , Animales , Células CHO , Estructuras de la Membrana Celular/genética , Cricetinae , Cricetulus , Perros , Proteínas Ligadas a GPI/genética , Células de Riñón Canino Madin DarbyRESUMEN
Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF-SRF (myocardin-related transcription factor-serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Entosis/fisiología , Transactivadores/metabolismo , Transcripción Genética/fisiología , Regulación hacia Arriba/fisiología , Línea Celular Tumoral , Estructuras de la Membrana Celular/genética , Proteínas del Citoesqueleto/genética , Humanos , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transactivadores/genéticaRESUMEN
T cell antigen receptor (TCR) stimulation induces recruitment and accumulation of various types of signaling molecules and forms signaling microclusters. The dynamics of the microclusters are important for regulating the quality and quantity of T cell activation. We describe here our protocols for analysis of signaling microclusters by using supported planar bilayers.
Asunto(s)
Estructuras de la Membrana Celular/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Estructuras de la Membrana Celular/genética , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genéticaRESUMEN
Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an epidermal growth factor (EGF)-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments, activating the Arp2/3 complex, and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating lamellipodia and ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium- and ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Estructuras de la Membrana Celular/metabolismo , Seudópodos/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células COS , Estructuras de la Membrana Celular/genética , Chlorocebus aethiops , Forminas , Células HeLa , Humanos , Seudópodos/genéticaRESUMEN
Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the correct time in the cell cycle. The structure and dynamics of this machine (divisome) in bacterial model systems are coming more clearly into focus, thanks to incisive cell biology methods in combination with biochemical and genetic approaches. The main conserved structural element of the machine is the tubulin homologue FtsZ, which assembles into a circumferential ring at the division site that is stabilized and anchored to the inner surface of the cytoplasmic membrane by FtsZ-binding proteins. Once this ring is in place, it recruits a series of transmembrane proteins that ultimately trigger cytokinesis. This review will survey the methods used to characterize the structure of the bacterial divisome, focusing mainly on the Escherichia coli model system, as well as the challenges that remain. These methods include recent super-resolution microscopy, cryo-electron tomography and synthetic reconstitution.
Asunto(s)
Bacterias/citología , Bacterias/metabolismo , División Celular/fisiología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular/genética , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/metabolismo , Citocinesis , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Mapeo de Interacción de ProteínasRESUMEN
RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.
Asunto(s)
ARN Helicasas DEAD-box/genética , Endorribonucleasas/genética , Proteínas de Escherichia coli/genética , Complejos Multienzimáticos/genética , Polirribonucleótido Nucleotidiltransferasa/genética , ARN Helicasas/genética , Estabilidad del ARN/genética , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/genética , ARN Helicasas DEAD-box/química , Endorribonucleasas/química , Escherichia coli/genética , Simulación de Dinámica Molecular , Complejos Multienzimáticos/química , Conformación de Ácido Nucleico , Fosfolípidos/química , Fosfolípidos/genética , Polirribonucleótido Nucleotidiltransferasa/química , Mapas de Interacción de Proteínas/genética , ARN Helicasas/química , ARN Mensajero/genéticaRESUMEN
Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.
Asunto(s)
Archamoebae/genética , Metabolismo Energético/genética , Evolución Molecular , Duplicación de Gen , Transferencia de Gen Horizontal , Orgánulos/genética , Anaerobiosis/genética , Archamoebae/enzimología , Archamoebae/metabolismo , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/metabolismo , Enzimas/genética , Enzimas/aislamiento & purificación , Orgánulos/enzimología , Orgánulos/metabolismoRESUMEN
ßArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that ßarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of ßarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of ßarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of ßarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that ßarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.
Asunto(s)
Arrestinas/metabolismo , Estructuras de la Membrana Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/biosíntesis , Animales , Arrestinas/genética , Estructuras de la Membrana Celular/genética , Movimiento Celular/fisiología , Activación Enzimática/fisiología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , beta-Arrestinas , Factores de Intercambio de Guanina Nucleótido ras/genéticaRESUMEN
Outer membrane vesicles (OMVs) derived from the alphaproteobacterium Ahrensia kielensis and from Pseudoalteromonas marina, a gammaproteobacterium, were sampled from liquid cultures in order to extract the MV-associated DNA, establish a shotgun library, and sequence randomly chosen clones to determine the origins of their DNA. We show that OMVs from A. kielensis and from P. marina both harbour DNA larger than 20 or 30 kbp. Transmission electron microscopical inspection of OMVs of A. kielensis and P. marina showed two types of vesicles: bilayered OMVs with a diameter between 30 and 250 nm and double bilayered OMVs ranging between 80 and 200 nm. Bilayered OMVs are either characterized by the presence of a large electron-dense substance or are elctron translucent. Double bilayered OMVs contained an electron dense substance in the core region surrounded by the second bilayer. 30,094 bp of the genome from OMV of A. kielensis and 45,981 bp of that from P. marina were sequenced. The results indicated that all sequences were single copy and that all sequences, with one exception, were similar to prokaryotic sequences, inserted viral sequences were not detected.
Asunto(s)
Alphaproteobacteria/fisiología , Estructuras de la Membrana Celular/ultraestructura , ADN Bacteriano/análisis , Pseudoalteromonas/fisiología , Alphaproteobacteria/genética , Alphaproteobacteria/ultraestructura , Proteínas de la Membrana Bacteriana Externa/química , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/fisiología , Pseudoalteromonas/genética , Pseudoalteromonas/ultraestructuraRESUMEN
Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Familia-src Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Aorta/metabolismo , Aorta/patología , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/patología , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Activación Enzimática/genética , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Mutación Missense , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Oxidación-Reducción , Unión Proteica , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal/genética , Porcinos , Dominios Homologos src , Familia-src Quinasas/genéticaRESUMEN
Insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) is the hallmark of glucose metabolism. In this study we examined insulin's effects on GLUT4 organization in PM of adipose cells by direct microscopic observation of single monomers tagged with photoswitchable fluorescent protein. In the basal state, after exocytotic delivery only a fraction of GLUT4 is dispersed into the PM as monomers, while most of the GLUT4 stays at the site of fusion and forms elongated clusters (60-240 nm). GLUT4 monomers outside clusters diffuse freely and do not aggregate with other monomers. In contrast, GLUT4 molecule collision with an existing cluster can lead to immediate confinement and association with that cluster. Insulin has three effects: it shifts the fraction of dispersed GLUT4 upon delivery, it augments the dissociation of GLUT4 monomers from clusters â¼3-fold and it decreases the rate of endocytic uptake. All together these three effects of insulin shift most of the PM GLUT4 from clustered to dispersed states. GLUT4 confinement in clusters represents a novel kinetic mechanism for insulin regulation of glucose homeostasis.
Asunto(s)
Adipocitos/metabolismo , Estructuras de la Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Homeostasis/fisiología , Insulina/metabolismo , Adipocitos/citología , Animales , Estructuras de la Membrana Celular/genética , Glucosa/genética , Transportador de Glucosa de Tipo 4/genética , Masculino , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.