RESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). Glial cells play roles in MN degeneration in ALS. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. To characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harboring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.
RESUMO
Amyotrophic lateral sclerosis (ALS) represents a complex neurodegenerative disorder with significant genetic heterogeneity. To date, both the genetic etiology and the underlying molecular mechanisms driving this disease remain poorly understood, although in recent years several studies have highlighted a number of genetic mutations causative for ALS. With these mutations pointing to potential pathways that may be affected within individuals with ALS, having the ability to generate human neurons and other disease relevant cells containing these mutations becomes even more critical if new therapies are to emerge. Recent developments with the advent of induced pluripotent stem cells (iPSCs) and clustered regularly interspaced short palindromic repeats (CRISPR) gene editing fields gave us the tools to introduce or correct a specific mutation at any site within the genome of an iPSC, and thus model the specific contribution of risk mutations. In this study we describe a rapid and efficient way to either introduce a mutation into a control line, or to correct an allele-specific mutation, generating an isogenic control line from patient-derived iPSCs with a given mutation. The mutations introduced were the G94A (also known as G93A) mutation into SOD1 or H517Q into FUS, and the mutation corrected was a patient iPSC line with I114T mutation in SOD1. A combination of small molecules and growth factors were used to guide a stepwise differentiation of the edited cells into motor neurons in order to demonstrate that disease-relevant cells could be generated for downstream applications. Through a combination of iPSCs and CRISPR editing, the cells generated here will provide fundamental insights into the molecular mechanisms underlying neuron degeneration in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Fluxo de TrabalhoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Superóxido Dismutase-1/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Anticorpos/imunologia , Linhagem Celular , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , NF-kappa B/metabolismo , Agregados Proteicos , Dobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Transgênicos , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/imunologia , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/genética , UbiquitinaçãoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of dominant-inherited Parkinson's disease (PD), and yet we do not fully understand the physiological function(s) of LRRK2. Various components of the clathrin machinery have been recently found mutated in familial forms of PD. Here, we provide molecular insight into the association of LRRK2 with the clathrin machinery. We report that through its GTPase domain, LRRK2 binds directly to clathrin-light chains (CLCs). Using genome-edited HA-LRRK2 cells, we localize LRRK2 to endosomes on the degradative pathway, where it partially co-localizes with CLCs. Knockdown of CLCs and/or LRRK2 enhances the activation of the small GTPase Rac1, leading to alterations in cell morphology, including the disruption of neuronal dendritic spines. In Drosphila, a minimal rough eye phenotype caused by overexpression of Rac1, is dramatically enhanced by loss of function of CLC and LRRK2 homologues, confirming the importance of this pathway in vivo. Our data identify a new pathway in which CLCs function with LRRK2 to control Rac1 activation on endosomes, providing a new link between the clathrin machinery, the cytoskeleton and PD.
Assuntos
Cadeias Leves de Clatrina/metabolismo , Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Encéfalo/citologia , Encéfalo/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Cadeias Leves de Clatrina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Olho/metabolismo , Olho/patologia , Técnicas de Silenciamento de Genes , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Dados de Sequência Molecular , Neurônios/metabolismo , Neurônios/patologia , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Ratos , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine-nucleotide exchange factors (GEFs), each Rab binds a specific set of effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase-activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase-activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas Ativadoras de GTPase/química , Humanos , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Troca de Nucleotídeo Guanina Rho/química , Proteínas rab de Ligação ao GTP/químicaRESUMO
Cells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to downregulate an Arf6-dependent recycling pathway for ß1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes of decreased cell adhesion and increased cell migration observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Células COS , Adesão Celular/genética , Movimento Celular/genética , Chlorocebus aethiops , Caderinas de Desmossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Integrina beta1/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in TARDBP (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9, we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease.
RESUMO
A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Fluxo de Trabalho , Neurônios Motores/metabolismoRESUMO
The v-SNARE TI-VAMP (VAMP7) mediates exocytosis during neuritogenesis, phagocytosis and lysosomal secretion. It localizes to endosomes and lysosomes but also to the trans-Golgi network. Here we show that depletion of TI-VAMP enhances the endocytosis of activated EGF receptor (EGFR) without affecting constitutive endocytosis of EGFR, or transferrin uptake. This increased EGFR internalization is mainly clathrin dependent. Searching for defects in EGFR regulators, we found that TI-VAMP depletion reduces the cell surface amount of CD82, a tetraspanin known to control EGFR localization in microdomains. We further show that TI-VAMP is required for secretion from the Golgi apparatus to the cell surface, and that TI-VAMP-positive vesicles transport CD82. Quantum dots video-microscopy indicates that depletion of TI-VAMP, or its cargo CD82, restrains EGFR diffusion and the area explored by EGFR at the cell surface. Both depletions also impair MAPK signaling and enhance endocytosis of activated EGFR by increased recruitment of AP-2. These results highlight the role of TI-VAMP in the secretory pathway of a tetraspanin, and support a model in which CD82 allows EGFR entry in microdomains that control its clathrin-dependent endocytosis and signaling.
Assuntos
Receptores ErbB/metabolismo , Proteína Kangai-1/metabolismo , Proteínas R-SNARE/metabolismo , Transdução de Sinais/fisiologia , Western Blotting , Clatrina/metabolismo , Endocitose/genética , Endocitose/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia Confocal , Proteínas R-SNARE/genética , Transdução de Sinais/genéticaRESUMO
Astrocytes play important roles in the function and survival of neuronal cells. Dysfunctions of astrocytes are associated with numerous disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Human-induced pluripotent stem cell (iPSC)-based approaches are becoming increasingly important for the study of the mechanisms underlying the involvement of astrocytes in non-cell autonomous processes of motor neuron degeneration in ALS. These studies must account for the molecular and functional diversity among astrocytes in different regions of the brain and spinal cord. It is essential that the most pathologically relevant astrocyte preparations are used when investigating non-cell autonomous mechanisms of either upper or lower motor neuron degeneration in ALS. Here, we describe the efficient and streamlined generation of human iPSC-derived astrocytes with molecular and biological properties similar to physiological astrocytes in the ventral spinal cord. These induced astrocytes exhibit spontaneous and ATP-induced calcium transients, and lack signs of overt activation. Human iPSC-derived astrocytes with ventral spinal cord features offer advantages over more generic astrocyte preparations for the study of both ventral spinal cord astrocyte biology and the involvement of astrocytes in mechanisms of lower motor neuron degeneration in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Esclerose Lateral Amiotrófica/patologia , Astrócitos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios Motores/patologia , Degeneração Neural/patologiaRESUMO
Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (â¼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.
RESUMO
Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Regulação Alostérica , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Autorrenovação Celular , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Invasividade Neoplásica , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Análise de SobrevidaRESUMO
Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.
Assuntos
Actinas/química , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Endocitose , Transferência Ressonante de Energia de Fluorescência , Lipossomos/química , Masculino , Camundongos , Camundongos Knockout , Ratos , Receptores de Superfície Celular/metabolismo , Células de Sertoli/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that selectively affects motor neurons (MNs) of the cortex, brainstem, and spinal cord. Several genes have been linked to both familial (fALS) and sporadic (sALS) cases of ALS. Among all the ALS-related genes, a group of genes known to directly affect cytoskeletal dynamics (ALS2, DCTN1, PFN1, KIF5A, NF-L, NF-H, PRPH, SPAST, and TUBA4A) is of high importance for MN health and survival, considering that MNs are large polarized cells with axons that can reach up to 1 m in length. In particular, cytoskeletal dynamics facilitate the transport of organelles and molecules across the long axonal distances within the cell, playing a key role in synapse maintenance. The majority of ALS-related genes affecting cytoskeletal dynamics were identified within the past two decades, making it a new area to explore for ALS. The purpose of this review is to provide insights into ALS-associated cytoskeletal genes and outline how recent studies have pointed towards novel pathways that might be impacted in ALS. Further studies making use of extensive analysis models to look for true hits, the newest technologies such as CRIPSR/Cas9, human induced pluripotent stem cells (iPSCs) and axon sequencing, as well as the development of more transgenic animal models could potentially help to: differentiate the variants that truly act as a primary cause of the disease from the ones that act as risk factors or disease modifiers, identify potential interactions between two or more ALS-related genes in disease onset and progression and increase our understanding of the molecular mechanisms leading to cytoskeletal defects. Altogether, this information will give us a hint on the real contribution of the cytoskeletal ALS-related genes during this lethal disease.
RESUMO
Astrocytes play a number of key functions in health and disease. Activated astrocytes are present in most, if not all, neurological diseases. Most current information on the mechanisms underlying reactive astrocyte emergence derives from studies using animal experimental systems, mainly because the ability to study human astrocytes under healthy and pathological conditions has been hampered by the difficulty in obtaining primary human astrocytes. Here we describe robust and reliable derivation of astrocytes from human induced pluripotent stem cells (iPSCs). Phenotypically characterized human iPSC-derived astrocytes exhibit typical traits of physiological astrocytes, including spontaneous and induced calcium transients. Moreover, human iPSC-derived astrocytes respond to stimulation with a pro-inflammatory combination of tumor necrosis factor-alpha, interleukin 1-alpha, and complement component C1q by undergoing changes in gene expression patterns suggesting acquisition of a reactive astrocyte phenotype. Together, these findings provide evidence suggesting that human iPSC-derived astrocytes are a suitable experimental model system to study astrocyte function and reactivation in healthy and pathological conditions of the human nervous system.
Assuntos
Astrócitos/metabolismo , Diferenciação Celular/fisiologia , Descoberta de Drogas , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas/métodos , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Neurodegenerative diseases are a challenge for drug discovery, as the biological mechanisms are complex and poorly understood, with a paucity of models that faithfully recapitulate these disorders. Recent advances in stem cell technology have provided a paradigm shift, providing researchers with tools to generate human induced pluripotent stem cells (iPSCs) from patient cells. With the potential to generate any human cell type, we can now generate human neurons and develop "first-of-their-kind" disease-relevant assays for small molecule screening. Now that the tools are in place, it is imperative that we accelerate discoveries from the bench to the clinic. Using traditional closed-door research systems raises barriers to discovery, by restricting access to cells, data and other research findings. Thus, a new strategy is required, and the Montreal Neurological Institute (MNI) and its partners are piloting an "Open Science" model. One signature initiative will be that the MNI biorepository will curate and disseminate patient samples in a more accessible manner through open transfer agreements. This feeds into the MNI open drug discovery platform, focused on developing industry-standard assays with iPSC-derived neurons. All cell lines, reagents and assay findings developed in this open fashion will be made available to academia and industry. By removing the obstacles many universities and companies face in distributing patient samples and assay results, our goal is to accelerate translational medical research and the development of new therapies for devastating neurodegenerative disorders.
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The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer-based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell's leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.
Assuntos
Movimento Celular , Neoplasias Epiteliais e Glandulares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Transporte Proteico/genética , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are the core machinery of membrane fusion. Vesicular SNAREs (v-SNAREs) interact with their target SNAREs (t-SNAREs) to form SNARE complexes which mediate membrane fusion. Here we review the basic properties and functions of the v-SNARE TI-VAMP/VAMP7 (Tetanus neurotoxin insensitive-vesicle associated membrane protein). TI-VAMP interacts with its t-SNARE partners, particularly plasmalemmal syntaxins, to mediate membrane fusion and with several regulatory proteins especially via its amino-terminal regulatory Longin domain. Partners include AP-3, Hrb/(Human immunodeficiency virus Rev binding) protein, and Varp (Vps9 domain and ankyrin repeats containing protein) and regulate TI-VAMP's function and targeting. TI-VAMP is involved both in secretory and endocytic pathways which mediate neurite outgrowth and synaptic transmission, plasma membrane remodeling and lysosomal secretion.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Metaloendopeptidases/metabolismo , Proteínas SNARE/metabolismo , Toxina Tetânica/metabolismo , Animais , Humanos , Transporte ProteicoRESUMO
Human immunodeficiency virus Rev-binding protein (HRB), also called human Rev-interacting protein (hRIP) or Rev/Rex activation domain binding (RAB) is a partner of the tyrosine kinase substrate EPS15, and it has been recovered in the AP-2 interactome. EPS15 and AP-2 are involved in endocytosis, but the function of HRB in this process is still unknown. Here we identified HRB as a partner of the vesicular SNARE tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) in yeast two-hybrid screens and using biochemical assays. In HeLa cells, HRB localized both in the nucleus and in the cytoplasm. In the cytoplasm, HRB colocalized with clathrin-, AP-2-, EPS15-, and transferrin receptor-containing vesicles. We did not see significant colocalization between HRB and TI-VAMP in HeLa cells, and we saw partial colocalization with green fluorescent protein-TI-VAMP in stably expressing Madin-Darby canine kidney cells. Nevertheless using a pHLuorin-tagged TI-VAMP construct, we found that HRB and TI-VAMP colocalize close to the plasma membrane after 5 min of anti-green fluorescent protein antibody uptake. These results suggest that TI-VAMP and HRB may interact only during the early stages of endocytosis. Furthermore uptake experiments followed by fluorescence-activated cell sorting showed that the endocytosis of fluorescent transferrin and pHLuorin-TI-VAMP is strongly reduced in HRB knockdown cells. Altogether these results suggest that HRB is involved in clathrin-dependent endocytosis and recruits TI-VAMP in this process.