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1.
EMBO J ; 37(2): 235-254, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29158324

RESUMO

Retromer is an endosomal multi-protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans-Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer-associated RAB7-specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer-dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin-mediated mitophagy.


Assuntos
Autofagossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
3.
PLoS Genet ; 10(3): e1004103, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24625543

RESUMO

Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.


Assuntos
Elementos de DNA Transponíveis/genética , Células Eucarióticas/metabolismo , Genoma Humano , Proteínas de Ligação a DNA/genética , Genes Transgênicos Suicidas/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Transposases/genética
4.
Matrix Biol ; 93: 60-78, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32450218

RESUMO

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding ß1 and ß3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal ß1/ß3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal ß1/ß3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.


Assuntos
Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neuroepiteliais/citologia , Proteína rhoA de Ligação ao GTP/metabolismo , Movimento Celular , Plasticidade Celular , Citoplasma/metabolismo , Desenvolvimento Embrionário , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Neuroepiteliais/metabolismo , Fenótipo
5.
J Cell Biol ; 218(9): 3019-3038, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31431476

RESUMO

Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer's role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid-sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid-sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Longevidade , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microdomínios da Membrana/genética , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
6.
J Cell Biol ; 216(11): 3677-3693, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-28935632

RESUMO

The retromer complex, which recycles the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), is thought to consist of a cargo-selective VPS26-VPS29-VPS35 trimer and a membrane-deforming subunit of sorting nexin (SNX)-Bin, Amphyphysin, and Rvs (BAR; SNX-BAR) proteins. In this study, we demonstrate that heterodimers of the SNX-BAR proteins, SNX1, SNX2, SNX5, and SNX6, are the cargo-selective elements that mediate the retrograde transport of CI-MPR from endosomes to the TGN independently of the core retromer trimer. Using quantitative proteomics, we also identify the IGF1R, among more potential cargo, as another SNX5 and SNX6 binding receptor that recycles through SNX-BAR heterodimers, but not via the retromer trimer, in a ligand- and activation-dependent manner. Overall, our data redefine the mechanics of retromer-based sorting and call into question whether retromer indeed functions as a complex of SNX-BAR proteins and the VPS26-VPS29-VPS35 trimer.


Assuntos
Receptor IGF Tipo 2/metabolismo , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sistemas CRISPR-Cas , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/genética , Nexinas de Classificação/química , Nexinas de Classificação/genética , Fatores de Tempo , Transfecção , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Rede trans-Golgi/metabolismo
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