RESUMO
Upon infection of host cells, Salmonella enterica serovar Typhimurium resides in a modified-endosomal compartment referred to as the Salmonella-containing vacuole (SCV). SCV biogenesis is driven by multiple effector proteins translocated through two type III secretion systems (T3SS-1 and T3SS-2). While many host proteins targeted by these effector proteins have been characterised, the role of host lipids in SCV dynamics remains poorly understood. Previous studies have shown that S. Typhimurium infection in macrophages leads to accumulation of intracellular cholesterol, some of which concentrates in and around SCVs; however, the underlying mechanisms remain unknown. Here, we show that S. Typhimurium utilises the T3SS-2 effector SseJ to downregulate expression of the host cholesterol transporter ABCA1 in macrophages, leading to a ~45% increase in cellular cholesterol. Mechanistically, SseJ activates a signalling cascade involving the host kinases FAK and Akt to suppress Abca1 expression. Mutational inactivation of SseJ acyltransferase activity, silencing FAK, or inhibiting Akt prevents Abca1 downregulation and the corresponding accumulation of cholesterol during infection. Importantly, RNAi-mediated silencing of ABCA1 rescued bacterial survival in FAK-deficient macrophages, suggesting that Abca1 downregulation and cholesterol accumulation are important for intracellular survival.
Assuntos
Proteínas de Transporte , Salmonella typhimurium , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Colesterol , Homeostase , Macrófagos/metabolismo , Salmonella typhimurium/metabolismoRESUMO
Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca(2+) channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Ebolavirus/fisiologia , Endossomos/virologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia , Modelos Biológicos , Proteína C1 de Niemann-PickRESUMO
UNLABELLED: Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE: Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.
Assuntos
Transporte Biológico , Ebolavirus/fisiologia , Endossomos/virologia , Lisossomos/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Internalização do Vírus , Proteínas de Transporte/análise , Linhagem Celular , Endossomos/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/química , Glicoproteínas de Membrana/análise , Proteína C1 de Niemann-Pick , Fatores de Tempo , Virossomos/metabolismoRESUMO
Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK(-/-) macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Macrófagos Peritoneais/imunologia , Fagocitose , Proteínas Proto-Oncogênicas c-akt/metabolismo , Salmonella typhimurium/imunologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Autofagia , Proteína 5 Relacionada à Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Ativação Enzimática , Escherichia coli/imunologia , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Quinase 1 de Adesão Focal/genética , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Viabilidade Microbiana , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Organismos Livres de Patógenos Específicos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast-two-hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP-22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Inflamação/microbiologia , Inflamação/patologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Salmonella typhimurium/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Genes Supressores de Tumor , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Migração Transendotelial e Transepitelial , Técnicas do Sistema de Duplo-HíbridoRESUMO
A FASEB Summer Research Conference entitled 'Arf and Rab family G proteins' was held in July 2013 at Snowmass Village, Snowmass, Colorado. Arfs and Rabs are two families of GTPases that control membrane trafficking in eukaryotic cells, and increasing evidence indicates that their functions are tightly coordinated. Because many workers in this field have focused on only one family, this meeting was designed to integrate our understanding of the two families. The conference was organized by Elizabeth Sztul (University of Alabama, Birmingham, USA) and Jim Casanova (University of Virginia, Charlottesville, USA), and provided an opportunity for approximately 90 scientists to communicate their work and discuss future directions for the field. The talks highlighted the structural, functional and regulatory properties of Arf and Rab GTPases and the need to develop coordinated approaches to investigate them. Here, we present the major themes that emerged from the meeting.
Assuntos
Fatores de Ribosilação do ADP/genética , Proteínas rab de Ligação ao GTP/genética , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Transdução de Sinais , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Integrin-mediated adhesion regulates membrane binding sites for Rac1 within lipid rafts. Detachment of cells from the substratum triggers the clearance of rafts from the plasma membrane through caveolin-dependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration, but the mechanisms are poorly understood. Here we show that endocytosis of rafts after detachment requires F-actin, followed by microtubule-dependent trafficking to recycling endosomes. When cells are replated on fibronectin, rafts exit from recycling endosomes in an Arf6-dependent manner and return to the plasma membrane along microtubules. Both of these steps are required for the plasma membrane targeting of Rac1 and for its activation. These data therefore define a new membrane raft trafficking pathway that is crucial for anchorage-dependent signalling.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Endocitose , Exocitose , Microdomínios da Membrana/fisiologia , Microtúbulos/fisiologia , Fator 6 de Ribosilação do ADP , Actinas/fisiologia , Animais , Adesão Celular , Forma Celular , Células Cultivadas , Retículo Endoplasmático/fisiologia , Fibroblastos/fisiologia , Fibronectinas/metabolismo , Complexo de Golgi/fisiologia , Camundongos , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.
Assuntos
Proteínas Angiogênicas/fisiologia , Aderência Bacteriana , Macrófagos/microbiologia , Salmonella typhimurium/fisiologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , CamundongosRESUMO
Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disabilities. BRAG1 is a guanine nucleotide exchange factor for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif; however, the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors, depresses AMPA receptor (AMPA-R)-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic XLID.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Região CA1 Hipocampal/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Glicoproteínas de Membrana/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores de AMPA/metabolismo , Sulfotransferases/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Motivos de Aminoácidos/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Calmodulina/metabolismo , Quelantes/farmacologia , Proteína 4 Homóloga a Disks-Large , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ionomicina/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Glicoproteínas de Membrana/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/fisiologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Ratos , Receptores de AMPA/genética , Sulfotransferases/genética , Transfecção , Valina/análogos & derivados , Valina/farmacologiaRESUMO
ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of ß1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrina alfa5beta1/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Substituição de Aminoácidos , Adesão Celular/fisiologia , Clatrina/genética , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Integrina alfa5beta1/genética , Mutação de Sentido IncorretoRESUMO
We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Hepacivirus/fisiologia , Replicação Viral , Fator 1 de Ribosilação do ADP/antagonistas & inibidores , Brefeldina A/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Hepatócitos/virologia , Humanos , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.
Assuntos
Fagossomos/enzimologia , Transporte Proteico/fisiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Lisossomos/enzimologia , Mutação , Salmonella typhimurium/genética , Vacúolos/enzimologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin.
Assuntos
Proteína rhoA de Ligação ao GTP , Ligação Proteica , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Animais , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Complexo de Golgi/metabolismo , Camundongos , Microtúbulos/metabolismo , Dinâmica Mitocondrial , Transdução de Sinais/genéticaRESUMO
Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.
Assuntos
Células Epiteliais/citologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Citoplasma/metabolismo , Cães , Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glutationa Transferase/metabolismo , Complexo de Golgi/metabolismo , Mutação , Transporte Proteico , Ratos , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The GTPase Arf6 regulates multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion, and cell migration [1, 2]. The Arf6-specific GAP ACAP1 is a negative regulator of Arf6-mediated signaling [3-7]. However, regulation of ACAP1- and Arf6-mediated signaling by other cellular proteins is not well understood. GULP/CED-6 is a phosphotyrosine binding (PTB)-domain-containing adaptor protein linked to engulfment of apoptotic cells [8-13] and to cholesterol homeostasis [14]. Here, we identify a novel role for GULP as a positive regulator of Arf6. Knockdown of GULP decreased cellular Arf6-GTP, whereas GULP overexpression increased cellular Arf6-GTP. At the mechanistic level, GULP influenced Arf6 at four levels. First, GULP bound directly to GDP-bound Arf6 via its PTB domain. Second, GULP associated with the Arf6-GAP ACAP1 at endogenous levels. Third, GULP reversed the Arf6-GTP decrease induced by ACAP1, and countered the ACAP1-mediated inhibition of cell migration. Fourth, GULP, ACAP1, and GDP-bound Arf6 were part of a tripartite complex, suggesting sequestration of ACAP1 as one mechanism of GULP action. Taken together, these data identify GULP as a modifier of cellular Arf6-GTP through regulation of ACAP1. Because PTB-domain-containing adaptor proteins influence endocytosis and trafficking of membrane proteins and cell migration [15, 16], our data support a model wherein PTB-domain-containing adaptor proteins regulate Arf family proteins.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Linhagem Celular , Cricetinae , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Camundongos , Estrutura Terciária de ProteínaRESUMO
Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação a Ácido Graxo/fisiologia , Adesões Focais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Fatores de Ribosilação do ADP/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Metabolismo dos Lipídeos , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/fisiologiaRESUMO
The intracellular gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium gains entry into nonphagocytic cells by manipulating the assembly of the host actin cytoskeleton. S. enterica serovar Typhimurium entry requires a functional type III secretion system, a conduit through which bacterial effector proteins are directly translocated into the host cytosol. We and others have previously reported the enhancement of tyrosine kinase activities during Salmonella serovar Typhimurium infection; however, neither specific kinases nor their targets have been well characterized. In this study, we investigated the roles of the cellular Abelson tyrosine kinase (c-Abl) and the related protein Arg in the context of serovar Typhimurium infection. We found that bacterial internalization was inhibited by more than 70% in cells lacking both c-Abl and Arg and that treatment of wild-type cells with a pharmaceutical inhibitor of the c-Abl kinase, STI571 (imatinib), reduced serovar Typhimurium invasion efficiency to a similar extent. Bacterial infection led to enhanced phosphorylation of two previously identified c-Abl substrates, the adaptor protein CT10 regulator of kinase (CrkII) and the Abelson-interacting protein Abi1, a component of the WAVE2 complex. Furthermore, overexpression of the nonphosphorylatable form of CrkII resulted in decreased invasion. Taken together, these findings indicate that c-Abl is activated during S. enterica serovar Typhimurium infection and that its phosphorylation of multiple downstream targets is functionally important in bacterial internalization.
Assuntos
Células Epiteliais/microbiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Salmonella typhi/patogenicidade , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Benzamidas , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Mesilato de Imatinib , Fosforilação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-crk/metabolismo , Pirimidinas/farmacologiaRESUMO
The small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including beta1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility. The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs). Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro, but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of beta1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of beta1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of beta1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Adesão Celular/fisiologia , Endocitose/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator 6 de Ribosilação do ADP , Primers do DNA , Citometria de Fluxo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Integrina beta1/metabolismo , Microscopia de Fluorescência , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Salmonella typhimurium colonizes the intestinal epithelium by injecting an array of effector proteins into host cells that induces phagocytic uptake of attached bacteria. However, the host molecules targeted by these effectors remain poorly defined. Here, we demonstrate that S. typhimurium induces formation of focal adhesion-like complexes at sites of bacterial attachment and that both focal adhesion kinase (FAK) and the scaffolding protein p130Cas are required for Salmonella uptake. Entry of Salmonella into FAK(-/-) cells is dramatically impaired and can be restored to control levels by expression of wild-type FAK. Surprisingly, reconstitution of bacterial internalization requires neither the kinase domain of FAK nor activation of c-Src, but does require a C-terminal PXXP motif through which FAK interacts with Cas. Infection of Cas(-/-) cells is also impaired, and reconstitution of invasiveness requires the central Cas YXXP repeat domain. The invasion defect in Cas(-/-) cells can be suppressed by overexpression of FAK, suggesting a functional link between FAK and Cas in the regulation of Salmonella invasion. Together, these findings reveal a novel role for focal adhesion proteins in the invasion of host cells by Salmonella.