RESUMO
Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis.
Assuntos
Proteínas do Citoesqueleto , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transdução de Sinais , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The tumor suppressor gene WWOX is localized in an unstable chromosomal region and its expression is decreased or absent in several types of cancer. A low expression of WWOX is associated with a poor prognosis in breast cancer (BC). It has recently been shown that WWOX contributes to genome stability through its role in the DNA damage response (DDR). In breast cancer cells, WWOX inhibits homologous recombination (HR), and thus promotes the repair of DNA double-stranded breaks (DSBs) by non-homologous end joining (NHEJ). The fine-tuning modulation of HR activity is crucial. Its under or overstimulation inducing genome alterations that can induce cancer. MERIT40 is a positive regulator of the DDR. This protein is indispensable for the function of the multi-protein complex BRCA1-A, which suppresses excessive HR activity. MERIT40 also recruits Tankyrase, a positive regulator of HR, to the DSBs to stimulate DNA repair. Here, we identified MERIT40 as a new molecular partner of WWOX. We demonstrated that WWOX inhibited excessive HR activity induced by overexpression of MERIT40. We showed that WWOX impaired the MERIT40-Tankyrase interaction preventing the role of the complex on DSBs. Furthermore, we found that MERIT40 is overexpressed in BC and that this overexpression is associated to a poor prognosis. These results strongly suggest that WWOX, through its interaction with MERIT40, prevents the deleterious impact of excessive HR on BC development by inhibiting MERIT40-Tankyrase association. This inhibitory effect of WWOX would oppose MERIT40-dependent BC development.
Assuntos
Neoplasias da Mama , Recombinação Homóloga , Feminino , Humanos , Neoplasias da Mama/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Tanquirases/genética , Tanquirases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/genética , Oxidorredutase com Domínios WW/metabolismoRESUMO
Skin aspartic acid protease (SASPase) is believed to be a key enzyme involved in filaggrin processing during epidermal terminal differentiation. Since little is known about the regulation of SASPase function, the aim of this study was to identify involved protein partners in the process. Yeast two hybrid analyses using SASPase as bait against a human reconstructed skin library identified that the N-terminal domain of filaggrin 2 binds to the N-terminal fragment of SASPase. This interaction was confirmed in reciprocal yeast two hybrid screens and by Surface Plasmon Resonance analyses. Immunohistochemical studies in human skin, using specific antibodies to SASPase and the N-terminal domain of filaggrin 2, showed that the two proteins partially co-localized to the stratum granulosum. In vitro enzymatic assays showed that the N-terminal domain of filaggrin 2 enhanced the autoactivation of SASPase to its 14 kDa active form. Taken together, the data suggest that the N-terminal domain of filaggrin 2 regulates the activation of SASPase that may be a key event upstream of filaggrin processing to natural moisturizing factors in the human epidermis.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas S100/metabolismo , Pele/metabolismo , Ácido Aspártico Endopeptidases/análise , Ativação Enzimática , Proteínas Filagrinas , Humanos , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteínas S100/análiseRESUMO
BACKGROUND: The WW domain-containing oxidoreductase (WWOX) gene, frequently altered in breast cancer, encodes a tumor suppressor whose function is mediated through its interactions with cancer-related proteins, such as the pro-apoptotic protein p73α. RESULTS: To better understand the involvement of WWOX in breast tumorigenesis, we performed a yeast two-hybrid screen and co-immunoprecipitation assays to identify novel partners of this protein. We characterized the vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) as a new regulator of WWOX. In breast cancer cells, VOPP1 sequestrates WWOX in lysosomes, impairs its ability to associate with p73α, and inhibits WWOX-dependent apoptosis. Overexpressed VOPP1 potentiates cellular transformation and enhances the growth of transplanted tumors in vivo. VOPP1 is overexpressed in breast tumors, especially in tumors that retain WWOX. Moreover, increased expression of VOPP1 is associated with reduced survival of patients with WWOX-positive, but not with WWOX-negative, tumors. CONCLUSIONS: These findings emphasize the importance of the sequestration of WWOX by VOPP1 in addition to WWOX loss in breast tumors and define VOPP1 as a novel oncogene promoting breast carcinogenesis by inhibiting the anti-tumoral effect of WWOX.
Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismoRESUMO
Coordination between membrane trafficking and actin polymerization is fundamental in cell migration, but a dynamic view of the underlying molecular mechanisms is still missing. The Rac1 GTPase controls actin polymerization at protrusions by interacting with its effector, the Wave regulatory complex (WRC). The exocyst complex, which functions in polarized exocytosis, has been involved in the regulation of cell motility. Here, we show a physical and functional connection between exocyst and WRC. Purified components of exocyst and WRC directly associate in vitro, and interactions interfaces are identified. The exocyst-WRC interaction is confirmed in cells by co-immunoprecipitation and is shown to occur independently of the Arp2/3 complex. Disruption of the exocyst-WRC interaction leads to impaired migration. By using time-lapse microscopy coupled to image correlation analysis, we visualized the trafficking of the WRC towards the front of the cell in nascent protrusions. The exocyst is necessary for WRC recruitment at the leading edge and for resulting cell edge movements. This direct link between the exocyst and WRC provides a new mechanistic insight into the spatio-temporal regulation of cell migration.
Assuntos
Movimento Celular , Extensões da Superfície Celular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Subunidades Proteicas/metabolismoRESUMO
SNAREs constitute the core machinery of intracellular membrane fusion, but vesicular SNAREs localize to specific compartments via largely unknown mechanisms. Here, we identified an interaction between VAMP7 and SNAP-47 using a proteomics approach. We found that SNAP-47 mainly localized to cytoplasm, the endoplasmic reticulum (ER), and ERGIC and could also shuttle between the cytoplasm and the nucleus. SNAP-47 preferentially interacted with the trans-Golgi network VAMP4 and post-Golgi VAMP7 and -8. SNAP-47 also interacted with ER and Golgi syntaxin 5 and with syntaxin 1 in the absence of Munc18a, when syntaxin 1 is retained in the ER. A C-terminally truncated SNAP-47 was impaired in interaction with VAMPs and affected their subcellular distribution. SNAP-47 silencing further shifted the subcellular localization of VAMP4 from the Golgi apparatus to the ER. WT and mutant SNAP-47 overexpression impaired VAMP7 exocytic activity. We conclude that SNAP-47 plays a role in the proper localization and function of a subset of VAMPs likely via regulation of their transport through the early secretory pathway.
Assuntos
Proteínas Q-SNARE/fisiologia , Proteínas R-SNARE/metabolismo , Animais , Cães , Células Madin Darby de Rim Canino , Transporte Proteico , Frações Subcelulares/metabolismoRESUMO
The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.
Assuntos
Reparo do DNA , Instabilidade Genômica , Proteínas de Transporte Vesicular/metabolismo , Fator 2 Ativador da Transcrição/análise , Fator 2 Ativador da Transcrição/metabolismo , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteína Beclina-1 , Linhagem Celular Tumoral , Exocitose , Deleção de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/análiseRESUMO
Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microtúbulos/genética , Morfogênese/genética , Proteínas Nucleares/genética , alfa Catenina/genética , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Polaridade Celular , Forma Celular , Cães , Embrião não Mamífero , Células Epiteliais/citologia , Vetores Genéticos , Humanos , Lentivirus/genética , Células Madin Darby de Rim Canino , Proteínas dos Microtúbulos/antagonistas & inibidores , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Oryzias , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , alfa Catenina/metabolismoRESUMO
UNLABELLED: The WW domain containing oxidoreductase (WWOX) has recently been shown to inhibit of the Wnt/ß-catenin pathway by preventing the nuclear import of disheveled 2 (DVL2) in human breast cancer cells. Here, it is revealed that WWOX also interacts with the BCL9-2, a cofactor of the Wnt/ß-catenin pathway, to enhance the activity of the ß-catenin-TCF/LEF (T-cell factor/lymphoid enhancer factors family) transcription factor complexes. By using both a luciferase assay in MCF-7 cells and a Xenopus secondary axis induction assay, it was demonstrated that WWOX inhibits the BCL9-2 function in Wnt/ß-catenin signaling. WWOX does not affect the BCL9-2-ß-catenin association and colocalizes with BCL9-2 and ß-catenin in the nucleus of the MCF-7 cells. Moreover, WWOX inhibits the ß-catenin-TCF1 interaction. Further examination found that HDAC3 associates with BCL9-2, enhances the inhibitory effect of WWOX on BCL9-2 transcriptional activity, and promotes the WWOX-BCL9-2 interaction, independent of its deacetylase activity. However, WWOX does not influence the HDAC3-BCL9-2 interaction. Altogether, these results strongly indicate that nuclear WWOX interacts with BCL9-2 associated with ß-catenin only when BCL9-2 is in complex with HDAC3 and inhibits its transcriptional activity, in part, by inhibiting the ß-catenin-TCF1 interaction. The promotion of the WWOX-BCL9-2 interaction by HDAC3, independent of its deacetylase activity, represents a new mechanism by which this HDAC inhibits transcription. IMPLICATIONS: The inhibition of the transcriptional activity of BCL9-2 by WWOX and HDAC3 constitutes a new molecular mechanism and provides new insight for a broad range of cancers.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Histona Desacetilases/genética , Oxirredutases/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Camundongos , Oxirredutases/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW , Xenopus , beta Catenina/metabolismoRESUMO
Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMPcontaining late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMPpositive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.
Assuntos
Exocitose , Proteínas dos Microfilamentos/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Endossomos/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Humanos , Metaloproteinase 14 da Matriz/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica/patologia , Metástase Neoplásica/ultraestrutura , Proteínas de Transporte Vesicular/metabolismoRESUMO
Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3(MEL-26)), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3(MEL-26)-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Katanina , Complexos Multiproteicos/metabolismo , FosforilaçãoRESUMO
Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer's Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to ß-amyloid in brains of patients suffering from Alzheimer's Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495-505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
RhoA, a small GTPase, regulates epithelial integrity and morphogenesis by controlling filamentous actin assembly and actomyosin contractility. Another important cytoskeletal regulator, Moesin (Moe), an ezrin, radixin, and moesin (ERM) protein, has the ability to bind to and organize cortical F-actin, as well as the ability to regulate RhoA activity. ERM proteins have previously been shown to interact with both RhoGEF (guanine nucleotide exchange factors) and RhoGAP (GTPase activating proteins), proteins that control the activation state of RhoA, but the functions of these interactions remain unclear. We demonstrate that Moe interacts with an unusual RhoGAP, Conundrum (Conu), and recruits it to the cell cortex to negatively regulate RhoA activity. In addition, we show that cortically localized Conu can promote cell proliferation and that this function requires RhoGAP activity. Surprisingly, Conu's ability to promote growth also appears dependent on increased Rac activity. Our results reveal a molecular mechanism by which ERM proteins control RhoA activity and suggest a novel linkage between the small GTPases RhoA and Rac in growth control.
Assuntos
Proliferação de Células , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Membrana/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Forma Celular , Sobrevivência Celular , Olho Composto de Artrópodes/metabolismo , Drosophila melanogaster/citologia , Células Epiteliais/fisiologia , Epitélio/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Discos Imaginais/metabolismo , Masculino , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte ProteicoRESUMO
The compartmental organization of eukaryotic cells is maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface.
Assuntos
Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas R-SNARE/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Nocodazol/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , Moduladores de Tubulina/farmacologiaRESUMO
The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais Proximais/citologia , Microvilosidades/metabolismo , Actinas/metabolismo , Animais , Células Epiteliais/metabolismo , Células LLC-PK1 , Domínios e Motivos de Interação entre Proteínas , Sus scrofa , SuínosAssuntos
Redes Reguladoras de Genes/fisiologia , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Transporte Biológico/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Espaço Intracelular/metabolismo , Modelos Biológicos , Saccharomyces cerevisiaeRESUMO
The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.
Assuntos
Movimento Celular/fisiologia , Proteínas Ativadoras de GTPase/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Regulação para Baixo , Ativação Enzimática , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Centro Organizador dos Microtúbulos/fisiologia , Centro Organizador dos Microtúbulos/ultraestrutura , Ratos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/fisiologiaRESUMO
Abscission is the least understood step of cytokinesis. It consists of the final cut of the intercellular bridge connecting the sister cells at the end of mitosis, and is thought to involve membrane trafficking as well as lipid and cytoskeleton remodelling. We previously identified the Rab35 GTPase as a regulator of a fast recycling endocytic pathway that is essential for post-furrowing cytokinesis stages. Here, we report that the phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) 5-phosphatase OCRL, which is mutated in Lowe syndrome patients, is an effector of the Rab35 GTPase in cytokinesis abscission. GTP-bound (active) Rab35 directly interacts with OCRL and controls its localization at the intercellular bridge. Depletion of Rab35 or OCRL inhibits cytokinesis abscission and is associated with local abnormal PtdIns(4,5)P2 and F-actin accumulation in the intercellular bridge. These division defects are also found in cell lines derived from Lowe patients and can be corrected by the addition of low doses of F-actin depolymerization drugs. Our data demonstrate that PtdIns(4,5)P2 hydrolysis is important for normal cytokinesis abscission to locally remodel the F-actin cytoskeleton in the intercellular bridge. They also reveal an unexpected role for the phosphatase OCRL in cell division and shed new light on the pleiotropic phenotypes associated with Lowe disease.
Assuntos
Actinas/metabolismo , Citocinese/fisiologia , Metabolismo dos Lipídeos , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citocinese/genética , Células HeLa , Humanos , Técnicas In Vitro , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.
Assuntos
Neoplasias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular , Transformação Celular Neoplásica , Células Cultivadas , Células HCT116 , Humanos , Imunidade Inata , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , TransfecçãoRESUMO
The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.