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1.
EMBO Rep ; 24(3): e56007, 2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36588479

RESUMO

Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.


Assuntos
Dictyostelium , Legionella pneumophila , Legionella , Vacúolos/metabolismo , Legionella/metabolismo , Dictyostelium/microbiologia , Fosfatidilinositóis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/metabolismo
2.
Cell Microbiol ; 23(5): e13318, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33583106

RESUMO

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.


Assuntos
Dictyostelium/fisiologia , Retículo Endoplasmático/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Legionella pneumophila/fisiologia , Proteínas de Protozoários/metabolismo , Vacúolos/microbiologia , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/microbiologia , Dictyostelium/ultraestrutura , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático Rugoso/microbiologia , Retículo Endoplasmático Rugoso/fisiologia , GTP Fosfo-Hidrolases/genética , Homeostase , Interações Hospedeiro-Patógeno , Legionella pneumophila/crescimento & desenvolvimento , Movimento , Muramidase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Protozoários/genética , Vacúolos/fisiologia
3.
J Biol Chem ; 295(21): 7391-7403, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32303638

RESUMO

The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.


Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Quinases/metabolismo , Febre Q/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Coxiella burnetii/genética , Proteínas Ativadoras de GTPase/genética , Humanos , Fosforilação , Proteínas Quinases/genética , Febre Q/genética , Vacúolos/genética , Vacúolos/microbiologia
4.
J Biol Chem ; 293(40): 15569-15580, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30131335

RESUMO

Secretion of bacterial signaling proteins and adaptation to the host, especially during infection, are processes that are often linked in pathogenic bacteria. The human pathogen Staphylococcus aureus is equipped with a large arsenal of immune-modulating factors, allowing it to either subvert the host immune response or to create permissive niches for its survival. Recently, we showed that one of the low-molecular-weight protein tyrosine phosphatases produced by S. aureus, PtpA, is secreted during growth. Here, we report that deletion of ptpA in S. aureus affects intramacrophage survival and infectivity. We also observed that PtpA is secreted during macrophage infection. Immunoprecipitation assays identified several host proteins as putative intracellular binding partners for PtpA, including coronin-1A, a cytoskeleton-associated protein that is implicated in a variety of cellular processes. Of note, we demonstrated that coronin-1A is phosphorylated on tyrosine residues upon S. aureus infection and that its phosphorylation profile is linked to PtpA expression. Our results confirm that PtpA has a critical role during infection as a bacterial effector protein that counteracts host defenses.


Assuntos
Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Proteínas dos Microfilamentos/genética , Proteínas Tirosina Fosfatases/genética , Infecções Estafilocócicas/genética , Staphylococcus aureus/genética , Animais , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Dictyostelium/genética , Dictyostelium/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/patogenicidade , Tirosina/metabolismo , Virulência
5.
J Cell Sci ; 129(12): 2354-67, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27170354

RESUMO

Proteins that contain Eps15 homology domains (EHDs) in their C-terminus are newly identified key regulators of endosomal membrane trafficking. Here, we show that D. discoideum contains a single EHD protein (referred to as EHD) that localizes to endosomal compartments and newly formed phagosomes. We provide the first evidence that EHD regulates phagosome maturation. Deletion of EHD results in defects in intraphagosomal proteolysis and acidification. These defects are linked to early delivery of lysosomal enzymes and fast retrieval of the vacuolar H(+)-ATPase in maturing phagosomes. We also demonstrate that EHD physically interacts with DymA. Our results indicate that EHD and DymA can associate independently with endomembranes, and yet they share identical kinetics in recruitment to phagosomes and release during phagosome maturation. Functional analysis of ehd(-), dymA(-) and double dymA(-)ehd(-) knockout strains indicate that DymA and EHD play non-redundant and independent functions in phagosome maturation. Finally, we show that the absence of EHD leads to increased tubulation of endosomes, indicating that EHD participates in the scission of endosomal tubules, as reported for DymA.


Assuntos
Dictyostelium/metabolismo , Dinaminas/metabolismo , Fagossomos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Mutação/genética , Ligação Proteica , Proteólise , Proteínas de Protozoários/química , Imagem com Lapso de Tempo
6.
J Cell Sci ; 127(Pt 21): 4702-13, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25189617

RESUMO

Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between post-lysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluid-phase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to post-lysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA(-) cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfA-dependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.


Assuntos
Dictyostelium/metabolismo , Dictyostelium/microbiologia , Legionella pneumophila/fisiologia , Lisossomos/metabolismo , Interações Hospedeiro-Patógeno
7.
J Cell Sci ; 126(Pt 3): 756-66, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23264736

RESUMO

ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization, instead of the six in human, enabling a more precise functional analysis. Genetic invalidation of acapA resulted in multinucleated cells with cytokinesis defects. Mutant acapA(-) cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A-GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that the ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA•GTP mutant in wild-type cells led to a significant reduction in filopodia length. Together, our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Dictyostelium/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Pseudópodes/metabolismo , Animais , Movimento Celular/genética , Citocinese/genética , Dictyostelium/enzimologia , Proteínas Ativadoras de GTPase/genética , Humanos , Mutação/genética , Pseudópodes/patologia , RNA Interferente Pequeno/genética , Transgenes/genética
8.
Elife ; 122023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37158597

RESUMO

The amoeba-resistant bacterium Legionella pneumophila causes Legionnaires' disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associated Legionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoeba Dictyostelium discoideum. Dually fluorescence-labeled D. discoideum producing LCV and LD markers revealed that Sey1 as well as the L. pneumophila T4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1 mutant D. discoideum indicated that Sey1 and GTP promote this process. Sey1 and the L. pneumophila fatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.


Assuntos
Dictyostelium , Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Legionella pneumophila/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Macrófagos/metabolismo , Dictyostelium/metabolismo , Gotículas Lipídicas/metabolismo , Vacúolos/metabolismo , Legionella/metabolismo , Doença dos Legionários/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
9.
Microbiol Spectr ; 11(3): e0106623, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37036353

RESUMO

Host metabolism reprogramming is a key feature of Mycobacterium tuberculosis (Mtb) infection that enables the survival of this pathogen within phagocytic cells and modulates the immune response facilitating the spread of the tuberculosis disease. Here, we demonstrate that a previously uncharacterized secreted protein from Mtb, Rv1813c, manipulates the host metabolism by targeting mitochondria. When expressed in eukaryotic cells, the protein is delivered to the mitochondrial intermembrane space and promotes the enhancement of host ATP production by boosting the oxidative phosphorylation metabolic pathway. Furthermore, the release of cytochrome c from mitochondria, an early apoptotic event in response to short-term oxidative stress, is delayed in Rv1813c-expressing cells. This study reveals a novel class of mitochondria targeting effectors from Mtb that might participate in host cell metabolic reprogramming and apoptosis control during Mtb infections. IMPORTANCE In this article, using a combination of techniques (bioinformatics, structural biology, and cell biology), we identified and characterized a new class of effectors present only in intracellular mycobacteria. These proteins specifically target host cell mitochondria when ectopically expressed in cells. We showed that one member of this family (Rv1813c) affects mitochondria metabolism in a way that might twist the immune response. This effector also inhibits the cytochrome c exit from mitochondria, suggesting that it might alter normal host cell apoptotic capacities, one of the first defenses of immune cells against Mtb infection.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/metabolismo , Citocromos c/metabolismo , Tuberculose/microbiologia , Metabolismo Energético , Mitocôndrias/metabolismo , Interações Hospedeiro-Patógeno
10.
Traffic ; 10(2): 161-71, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19054384

RESUMO

Sorting of ubiquitinated proteins to multivesicular bodies (MVBs) in mammalian cells relies on proteins with a Vps27/Hrs/STAM (VHS) domain. Here, we show that the amoeba Dictyostelium presents only one protein with a VHS domain: DdTom1. We demonstrate that the VHS domain of DdTom1 is followed by a Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding and Tom1 (GAT) domain that binds ubiquitin, and by a non-conserved C-terminal domain that can recruit clathrin, EGFr pathway substrate 15 and tumor susceptibility gene 101, a component of the MVB biogenesis machinery [endosomal complexes required for transport (ESCRT) complexes]. Both VHS and GAT domains interact with phospholipids and therefore could ensure the recruitment of DdTom1 to endosomal membranes. We propose that DdTom1 participates in an ancestral ESCRT-0 complex implicated in the sorting of ubiquitinated proteins into MVBs.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Dictyostelium/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Animais , Clatrina/metabolismo , Citoplasma/metabolismo , Dictyostelium/genética , Dictyostelium/ultraestrutura , Deleção de Genes , Humanos , Lisossomos/metabolismo , Microscopia Eletrônica , Ligação Proteica , Proteínas de Protozoários/genética
11.
Front Cell Dev Biol ; 9: 731964, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34746129

RESUMO

The haploid social amoeba Dictyostelium discoideum is a powerful model organism to study vesicle trafficking, motility and migration, cell division, developmental processes, and host cell-pathogen interactions. Dynamin superfamily proteins (DSPs) are large GTPases, which promote membrane fission and fusion, as well as membrane-independent cellular processes. Accordingly, DSPs play crucial roles for vesicle biogenesis and transport, organelle homeostasis, cytokinesis and cell-autonomous immunity. Major progress has been made over the last years in elucidating the function and structure of mammalian DSPs. D. discoideum produces at least eight DSPs, which are involved in membrane dynamics and other processes. The function and structure of these large GTPases has not been fully explored, despite the elaborate genetic and cell biological tools available for D. discoideum. In this review, we focus on the current knowledge about mammalian and D. discoideum DSPs, and we advocate the use of the genetically tractable amoeba to further study the role of DSPs in cell and infection biology. Particular emphasis is put on the virulence mechanisms of the facultative intracellular bacterium Legionella pneumophila.

12.
Dev Cell ; 5(3): 499-511, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12967568

RESUMO

PtdIns(3,5)P(2) is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P(2) effector localized to endosomes. The ENTH domain of Ent3p is essential for its PtdIns(3,5)P(2) binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P(2) binding via its ENTH domain.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas , Neuropeptídeos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Alquil e Aril Transferases/metabolismo , Western Blotting , Clatrina/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas de Fluorescência Verde , Técnicas In Vitro , Cinética , Proteínas Luminescentes/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Mutação , Testes de Precipitina , Transporte Proteico , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Alinhamento de Sequência/métodos , Temperatura , Fatores de Tempo , Leveduras
13.
Exp Cell Res ; 314(15): 2822-33, 2008 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-18634783

RESUMO

Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Dictyostelium/metabolismo , Endocitose/fisiologia , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Fluorescência Verde/genética , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Substâncias Macromoleculares/metabolismo , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Vesículas Transportadoras/ultraestrutura
14.
Mol Biol Cell ; 17(12): 4982-7, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16987957

RESUMO

Dictyostelium amoebae grow as single cells but upon starvation they initiate multicellular development. Phg2 was characterized previously as a kinase controlling cellular adhesion and the organization of the actin cytoskeleton. Here we report that Phg2 also plays a role during the transition between growth and multicellular development, as evidenced by the fact that phg2 mutant cells can initiate development even in the presence of nutrients. Even at low cell density and in rich medium, phg2 mutant cells express discoidin, one of the earliest predevelopmental markers. Complementation studies indicate that, in addition to the kinase domain, the core region of Phg2 is involved in the initiation of development. In this region, a small domain contiguous with a previously described ras-binding domain was found to interact with the Dictyostelium ortholog of the mammalian adhesion-regulating molecule (ADRM1). In addition, adrm1 knockout cells also exhibit abnormal initiation of development. These results suggest that a Phg2-Adrm1 signaling pathway is involved in the control of the transition from growth to differentiation in Dictyostelium. Phg2 thus plays a dual role in the control of cellular adhesion and initiation of development.


Assuntos
Dictyostelium/crescimento & desenvolvimento , Alimentos , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Dictyostelium/citologia , Privação de Alimentos/fisiologia , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína
15.
ScientificWorldJournal ; 9: 629-32, 2009 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-19618090

RESUMO

Syndecans are transmembrane proteoglycan receptors that interact with a wide variety of extracellular molecules such as adhesion receptors and extracellular matrix components. There are four syndecans in mammals, which are expressed in a development-, cell-type-, and tissue-specific manner, and function either as coreceptors that cooperate with other cell surface receptors or as cell adhesion receptors that independently mediate cell signaling. Cell signaling is supported through their short cytoplasmic tail that contains four tyrosine residues, which are conserved among all syndecan family members. In this commentary, we report and discuss data showing that the receptor syndecan-1 interacts with the carboxy-terminal LG4/5 domain in laminin-332 to participate in cell adhesion and spreading. Remarkably, cell adhesion to LG4/5 is associated with a rapid dephosphorylation of tyrosine in syndecan-1. These results unveil for the first time that one "turn on" signal for syndecan-1 upon LG4/5 recognition may not be phosphorylation, but tyrosine dephosphorylation, an unexpected outcome. How this regulatory event may take place and which tyrosine residues are concerned are questions tackled in this report.


Assuntos
Sindecana-1/metabolismo , Tirosina/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Calinina
16.
J Cell Physiol ; 214(1): 238-49, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17579341

RESUMO

Laminin 5/laminin 332 (LN332) is an adhesion substrate for epithelial cells. After secretion of LN332, a regulated cleavage occurs at the carboxy-terminus of its alpha3 subunit, which releases a tandem of two globular modules named LG4/5. We show that the presence of the LG4/5 domain in precursor LN332 decreases its integrin-mediated cell adhesion properties in comparison with mature LN332. Whereas cell adhesion to the recombinant LG4/5 fragment relies solely on the heparan sulfate proteoglycan (HSPG) receptor syndecan-1, we reveal that both syndecan-1 and the alpha3beta1 integrin bind to precursor LN332. We further demonstrate that syndecan-1 mediated cell adhesion to the LG4/5 fragment and pre-LN332 allows the formation of fascin-containing protrusions, depending on the GTPases Rac and Cdc42 activation. Reducing syndecan-1 expression in normal keratinocytes prevents cell protrusions on pre-LN332 with subsequent failure of the peripheral localization of the alpha3beta1 integrin. We finally show that cell migration on pre-LN332 requires syndecan-1. Therefore, the LG4/5 domain in precursor LN332 appears to trigger intracellular signaling events, which participate in keratinocyte motility.


Assuntos
Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Queratinócitos/fisiologia , Sindecana-1/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/isolamento & purificação , Células Cultivadas , Ativação Enzimática , Fibrossarcoma/patologia , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Integrina alfa3beta1/metabolismo , Masculino , Melanoma/patologia , Microscopia de Vídeo , Faloidina , Plasmídeos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodaminas , Pele/citologia , Transfecção , Proteína cdc42 de Ligação ao GTP/análise , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/análise , Proteínas rac de Ligação ao GTP/metabolismo , Calinina
17.
Mol Biol Cell ; 15(7): 3031-41, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15107463

RESUMO

At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P(2) and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P(2) via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P(2)- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Endossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/análise , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Carboxipeptidases/análise , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/imunologia , Imunoprecipitação , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/análise , Alinhamento de Sequência , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/análise
18.
Mol Biol Cell ; 14(7): 2890-9, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12857872

RESUMO

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.


Assuntos
Dictyostelium/metabolismo , Proteínas de Membrana/metabolismo , Fagocitose/fisiologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Dictyostelium/crescimento & desenvolvimento , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Proteínas de Protozoários/fisiologia , Homologia de Sequência de Aminoácidos
19.
Mol Biol Cell ; 15(8): 3915-25, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15194808

RESUMO

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Dictyostelium/enzimologia , Dictyostelium/ultraestrutura , Proteínas Serina-Treonina Quinases/fisiologia , Sequência de Aminoácidos , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Forma Celular/genética , Forma Celular/fisiologia , Citocinese/genética , Citocinese/fisiologia , Dictyostelium/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação/genética , Miosinas/genética , Miosinas/fisiologia , Fagocitose/genética , Fagocitose/fisiologia , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Talina/genética , Talina/fisiologia
20.
Mol Biol Cell ; 14(5): 1835-51, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12802059

RESUMO

Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The gamma-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing gamma-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1-coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.


Assuntos
Complexo 1 de Proteínas Adaptadoras/genética , Dictyostelium/genética , Enzimas/metabolismo , Lisossomos/enzimologia , Vacúolos/metabolismo , Complexo 1 de Proteínas Adaptadoras/fisiologia , Sequência de Aminoácidos , Animais , Clatrina/metabolismo , Dictyostelium/fisiologia , Genes Reporter , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Transporte Proteico/fisiologia , Alinhamento de Sequência , Vacúolos/ultraestrutura
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