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1.
J Cell Sci ; 130(15): 2579-2590, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600323

RESUMO

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.


Assuntos
Endossomos/metabolismo , Lipoilação , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endossomos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/genética
2.
Mol Biol Cell ; 2016 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-27654944

RESUMO

Epidermal Growth Factor Receptor (EGFR) signaling is essential for animal development and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus-end directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell, genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants consistent with mammalian cell studies whereby dynein has been shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking.

3.
PLoS Genet ; 10(10): e1004728, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329472

RESUMO

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vulva/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Receptores ErbB/genética , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Genes Supressores , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Mucosa Intestinal/metabolismo , Larva/metabolismo , Lisossomos/genética , Lisossomos/patologia , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Vulva/citologia , Vulva/metabolismo
4.
PLoS One ; 7(4): e36489, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558469

RESUMO

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Receptores ErbB/metabolismo , Transdução de Sinais , Vulva/crescimento & desenvolvimento , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Feminino , Fenótipo , Transporte Proteico , Deleção de Sequência , Tela Subcutânea/metabolismo , Vulva/citologia , Vulva/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genética , Proteínas ras/metabolismo
5.
Opt Express ; 20(5): 5344-55, 2012 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-22418342

RESUMO

We propose for the first time an E. coli bacteria sensor based on the evanescent field of the fundamental mode of a suspended-core terahertz fiber. The sensor is capable of E. coli detection at concentrations in the range of 10(4)-10(9) cfu/ml. The polyethylene fiber features a 150 µm core suspended by three deeply sub-wavelength bridges in the center of a 5.1 mm-diameter cladding tube. The fiber core is biofunctionalized with T4 bacteriophages which bind and eventually destroy (lyse) their bacterial target. Using environmental SEM we demonstrate that E. coli is first captured by the phages on the fiber surface. After 25 minutes, most of the bacteria is infected by phages and then destroyed with ~1 µm-size fragments remaining bound to the fiber surface. The bacteria-binding and subsequent lysis unambiguously correlate with a strong increase of the fiber absorption. This signal allows the detection and quantification of bacteria concentration. Presented bacteria detection method is label-free and it does not rely on the presence of any bacterial "fingerprint" features in the THz spectrum.


Assuntos
Bacteriófago T4/fisiologia , Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Escherichia coli/isolamento & purificação , Escherichia coli/virologia , Tecnologia de Fibra Óptica/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Contagem de Colônia Microbiana/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Radiação Terahertz
6.
PLoS One ; 5(12): e15662, 2010 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-21203392

RESUMO

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Mutação , Alelos , Aminas/farmacologia , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Colesterol/metabolismo , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Fenótipo , Ligação Proteica
7.
J Biomed Opt ; 13(5): 054003, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19021383

RESUMO

Biodegradable microstructured polymer optical fibers have been created using synthetic biomaterials such as poly(L-lactic acid), poly(epsilon-caprolactone), and cellulose derivatives. Original processing techniques were utilized to fabricate a variety of biofriendly microstructured fibers that hold potential for in vivo light delivery, sensing, and controlled drug-release.


Assuntos
Implantes Absorvíveis , Materiais Biocompatíveis/química , Análise de Falha de Equipamento/métodos , Fibras Ópticas , Desenho de Equipamento , Teste de Materiais
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