RESUMO
Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a disease-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with WT CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.
Assuntos
Caveolina 1 , Caveolinas , Doença , Humanos , Cavéolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Subunidades Proteicas/metabolismo , Doença/genéticaRESUMO
Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer's disease. The cleavage of APP by ß-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-ß (Aß) peptides are essential steps in this pathway. Biochemical evidence suggests that amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid-ordered phase membrane rafts. However, direct evidence that C99 preferentially associates with these rafts has remained elusive. Here, we tested this by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles (GPMVs). We found that C99 was essentially excluded from ordered domains in vesicles from HeLa cells, undifferentiated SH-SY5Y cells, or SH-SY5Y-derived neurons; instead, â¼90% of C99 partitioned into disordered domains. The strong association of C99 with disordered domains occurred independently of its cholesterol-binding activity or homodimerization, or of the presence of the familial Alzheimer disease Arctic mutation (APP E693G). Finally, through biochemical studies we confirmed previous results, which showed that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying that either i) amyloidogenic processing of the protein occurs in disordered regions of the membrane, ii) processing involves a marginal subpopulation of C99 found in rafts, or iii) as-yet-unidentified protein-protein interactions with C99 in living cells drive this protein into membrane rafts to promote its cleavage therein.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Membrana Celular/química , Células HeLa , Humanos , Mutação , Domínios ProteicosRESUMO
Caveolin-1 (Cav1) is the primary scaffolding protein of caveolae, flask-shaped invaginations of the plasma membrane thought to function in endocytosis, mechanotransduction, signaling and lipid homeostasis. A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins. However, how different tags affect the behavior of ectopically expressed Cav1 is still largely unknown. To address this question, we performed a comparative analysis of the subcellular distribution, oligomerization state and detergent resistance of transiently overexpressed Cav1 labeled with three different C-terminal tags (EGFP, mCherry and myc). We show that addition of fluorescent protein tags enhances the aggregation and/or degradation of both wild-type Cav1 and an oligomerization defective P132L mutant. Strikingly, complexes formed by overexpressed Cav1 fusion proteins excluded endogenous Cav1 and Cav2, and the properties of native caveolins were largely preserved even when abnormal aggregates were present in cells. These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae. They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.
Assuntos
Caveolina 1/metabolismo , Animais , Células COS , Caveolina 2/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Chlorocebus aethiops , Endocitose/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Vermelha FluorescenteRESUMO
How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis.
Assuntos
Membrana Celular/metabolismo , Endocitose , Microtúbulos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Toxina da Cólera/metabolismo , Clatrina/metabolismo , Dineínas/metabolismo , Células HeLa , Humanos , Ligação Proteica , Receptores da Transferrina/metabolismoRESUMO
Vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed in endothelial cells and regulates angiogenic signal transduction under both physiological and pathological conditions. VEGFR2 turnover at the plasma membrane (PM) is regulated by its transport through endocytic and secretory transport pathways. Short-range cargo trafficking along actin filaments is commonly regulated by motor proteins of myosin superfamily. In the current study, performed in primary human endothelial cells, we demonstrate that unconventional myosin 1c (Myo1c; class I family member) regulates the localization of VEGFR2 at the PM. We further demonstrate that the recruitment of VEGFR2 to the PM and its colocalization with Myo1c and caveolin-1 occur in response to VEGF-A (VEGF) stimulation. In addition, VEGF-induced delivery of VEGFR2 to the cell surface requires Myo1c; surface VEGFR2 levels are reduced in the absence of Myo1c and, more importantly, are restored by the overexpression of wild-type but not mutant Myo1c. Subcellular density gradient fractionation revealed that partitioning of VEGFR2 into caveolin-1- and Myo1c-enriched membrane fractions is dependent on VEGF stimulation. Myo1c depletion resulted in increased VEGF-induced VEGFR2 transport to the lysosomes for degradation and was rescued by applying either brefeldin A, which blocks trafficking between the endoplasmic reticulum and the Golgi complex, or dynasore, an inhibitor of dynamin-mediated endocytosis. Myo1c depletion also reduced VEGF-induced VEGFR2 phosphorylation at Y1175 and phosphorylation-dependent activation of ERK1/2 and c-Src kinase, leading to reduced cell proliferation and cell migration. This is the first report demonstrating that Myo1c is an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling.
Assuntos
Células Endoteliais/metabolismo , Proteínas Motores Moleculares/metabolismo , Miosina Tipo I/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Antimaláricos/farmacologia , Brefeldina A/farmacologia , Caveolina 1/metabolismo , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Cloroquina/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrazonas/farmacologia , Microdomínios da Membrana/metabolismo , Proteínas Motores Moleculares/genética , Miosina Tipo I/genética , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Mensageiro/metabolismo , Via Secretória/efeitos dos fármacos , Via Secretória/fisiologiaRESUMO
Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent.
Assuntos
Complexo de Golgi/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Qa-SNARE/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/fisiologia , Proteínas SNARE/antagonistas & inibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Transfecção , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
IL (interleukin)-6, an established growth factor for multiple myeloma cells, induces myeloma therapy resistance, but the resistance mechanisms remain unclear. The present study determines the role of IL-6 in re-establishing intracellular redox homoeostasis in the context of myeloma therapy. IL-6 treatment increased myeloma cell resistance to agents that induce oxidative stress, including IR (ionizing radiation) and Dex (dexamethasone). Relative to IR alone, myeloma cells treated with IL-6 plus IR demonstrated reduced annexin/propidium iodide staining, caspase 3 activation, PARP [poly(ADP-ribose) polymerase] cleavage and mitochondrial membrane depolarization with increased clonogenic survival. IL-6 combined with IR or Dex increased early intracellular pro-oxidant levels that were causally related to activation of NF-κB (nuclear factor κB) as determined by the ability of N-acetylcysteine to suppress both pro-oxidant levels and NF-κB activation. In myeloma cells, upon combination with hydrogen peroxide treatment, relative to TNF (tumour necrosis factor)-α, IL-6 induced an early perturbation in reduced glutathione level and increased NF-κB-dependent MnSOD (manganese superoxide dismutase) expression. Furthermore, knockdown of MnSOD suppressed the IL-6-induced myeloma cell resistance to radiation. MitoSOX Red staining showed that IL-6 treatment attenuated late mitochondrial oxidant production in irradiated myeloma cells. The present study provides evidence that increases in MnSOD expression mediate IL-6-induced resistance to Dex and radiation in myeloma cells. The results of the present study indicate that inhibition of antioxidant pathways could enhance myeloma cell responses to radiotherapy and/or chemotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Interleucina-6/fisiologia , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/terapia , Estresse Oxidativo/fisiologia , Superóxido Dismutase/biossíntese , Regulação para Cima/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Mieloma Múltiplo/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismoRESUMO
The α5ß1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5ß1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5ß1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of ß1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5ß1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Fibronectinas , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Proteínas Qa-SNARE/metabolismo , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Endossomos/metabolismo , Células Endoteliais/citologia , Ativação Enzimática/fisiologia , Humanos , Lisossomos/metabolismo , Transporte Proteico/fisiologia , Proteólise , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Sheath blight caused by necrotrophic fungus Rhizoctonia solani Kühn is one of the most serious diseases of rice. Use of high yielding semi dwarf cultivars with dense planting and high dose of nitrogenous fertilizers accentuates the incidence of sheath blight in rice. Its diverse host range and ability to remain dormant under unfavorable conditions make the pathogen more difficult to manage. As there are no sources of complete resistance, management through chemical control has been the most adopted method for sheath blight management. In this review, we provide an up-to-date comprehensive description of host-pathogen interactions, various control measures such as cultural, chemical, and biological as well as utilizing host plant resistance. The section on utilizing host plant resistance includes identification of resistant sources, mapping QTLs and their validation, identification of candidate gene(s) and their introgression through marker-assisted selection. Advances and prospects of sheath blight management through biotechnological approaches such as overexpression of genes and gene silencing for transgenic development against R. solani are also discussed.
RESUMO
Plasma membrane organization profoundly impacts cellular functionality. A well-known mechanism underlying this organization is through nanoscopic clustering of distinct lipids and proteins in membrane rafts. Despite their physiological importance, rafts remain a difficult-to-study aspect of membrane organization, in part because of the paucity of chemical tools to experimentally modulate their properties. Methods to selectively target rafts for therapeutic purposes are also currently lacking. To tackle these problems, we developed a high-throughput screen and an accompanying image analysis pipeline to identify small molecules that enhance or inhibit raft formation. Cell-derived giant plasma membrane vesicles were used as the experimental platform. A proof-of-principle screen using a bioactive lipid library demonstrates that this method is robust and capable of validating established raft modulators including C6- and C8-ceramide, miltefosine, and epigallocatechin gallate as well as identifying new ones. The platform we describe here represents a powerful tool to discover new chemical approaches to manipulate rafts and their components.
RESUMO
Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.
Assuntos
Toxina da Cólera/metabolismo , Receptores de Superfície Celular/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , EndocitoseRESUMO
In the original version of this Article, the fifth sentence of the abstract incorrectly read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.', and should have read 'Remarkably, we show that PACSIN1 and EHD1 assemble membrane tubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell.'. This has been corrected in both the PDF and HTML versions of the Article.
RESUMO
The intracellular ciliogenesis pathway requires membrane trafficking, fusion, and reorganization. Here, we demonstrate in human cells and zebrafish that the F-BAR domain containing proteins PACSIN1 and -2 play an essential role in ciliogenesis, similar to their binding partner and membrane reorganizer EHD1. In mature cilia, PACSINs and EHDs are dynamically localized to the ciliary pocket membrane (CPM) and transported away from this structure on membrane tubules along with proteins that exit the cilium. PACSINs function early in ciliogenesis at the ciliary vesicle (CV) stage to promote mother centriole to basal body transition. Remarkably, we show that PACSIN1 and EHD1 assemble membrane t7ubules from the developing intracellular cilium that attach to the plasma membrane, creating an extracellular membrane channel (EMC) to the outside of the cell. Together, our work uncovers a function for F-BAR proteins and membrane tubulation in ciliogenesis and explains how the intracellular cilium emerges from the cell.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Corpos Basais/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Corpos Basais/ultraestrutura , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/ultraestrutura , Embrião não Mamífero , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Fusão de Membrana , Camundongos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Malaria parasite secretes various proteins in infected RBC for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine/drug against malaria. The existing motif-based methods have got limited success due to lack of universal motif in all secretory proteins of malaria parasite. RESULTS: In this study a systematic attempt has been made to develop a general method for predicting secretory proteins of malaria parasite. All models were trained and tested on a non-redundant dataset of 252 secretory and 252 non-secretory proteins. We developed SVM models and achieved maximum MCC 0.72 with 85.65% accuracy and MCC 0.74 with 86.45% accuracy using amino acid and dipeptide composition respectively. SVM models were developed using split-amino acid and split-dipeptide composition and achieved maximum MCC 0.74 with 86.40% accuracy and MCC 0.77 with accuracy 88.22% respectively. In this study, for the first time PSSM profiles obtained from PSI-BLAST, have been used for predicting secretory proteins. We achieved maximum MCC 0.86 with 92.66% accuracy using PSSM based SVM model. All models developed in this study were evaluated using 5-fold cross-validation technique. CONCLUSION: This study demonstrates that secretory proteins have different residue composition than non-secretory proteins. Thus, it is possible to predict secretory proteins from its residue composition-using machine learning technique. The multiple sequence alignment provides more information than sequence itself. Thus performance of method based on PSSM profile is more accurate than method based on sequence composition. A web server PSEApred has been developed for predicting secretory proteins of malaria parasites,the URL can be found in the Availability and requirements section.
Assuntos
Inteligência Artificial , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Eritrócitos/parasitologia , Perfilação da Expressão Gênica/métodos , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Proteoma/metabolismo , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Eritrócitos/metabolismo , Humanos , Dados de Sequência MolecularRESUMO
Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting ß-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased ß-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote ß-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Clatrina/metabolismo , Drosophila melanogaster/metabolismo , Endocitose/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Humanos , Lactente , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Via de Sinalização WntRESUMO
Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1-/- MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1-/- MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.
Assuntos
Caveolina 1/genética , Caveolina 1/metabolismo , Cavéolas/fisiologia , Endocitose , Retículo Endoplasmático , Fibroblastos/metabolismo , Mutação da Fase de Leitura/genética , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Mutação , Transporte ProteicoRESUMO
In addition to containing highly dynamic nanoscale domains, the plasma membranes of many cell types are decorated with caveolae, flask-shaped domains enriched in the structural protein caveolin-1 (Cav1). The importance of caveolae in numerous cellular functions and processes has become well-recognized, and recent years have seen dramatic advances in our understanding of how caveolae assemble and the mechanisms control the turnover of Cav1. At the same time, work from our lab and others have revealed that commonly utilized strategies such as overexpression and tagging of Cav1 have unexpectedly complex consequences on the trafficking and fate of Cav1. Here, we discuss the implications of these findings for current models of caveolae biogenesis and Cav1 turnover. In addition, we discuss how disease-associated mutants of Cav1 impact caveolae assembly and outline open questions in this still-emerging area.
RESUMO
Caveolin-1 (Cav1) drives the formation of flask-shaped membrane invaginations known as caveolae that participate in signaling, clathrin-independent endocytosis and mechanotransduction. Overexpression or mutations of Cav1 can lead to its mistrafficking, including its accumulation in a perinuclear compartment previously identified as the Golgi complex. Here, we show that in the case of overexpressed Cav1-GFP, this perinuclear compartment consists of cytoplasmic inclusion bodies generated in response to the accumulation of aggregates of misfolded proteins, known as aggresomes. Aggresomes containing Cav1-GFP are encased within vimentin cages, form in a microtubule-dependent manner, and are enriched in a number of key regulators of protein turnover, including ubiquitin, VCP/p97 and proteasomes. Interestingly, aggresome induction was cell-type dependent and was observed for many but not all Cav1 constructs tested. Furthermore, endogenous Cav1 accumulated in aggresomes formed in response to proteosomal inhibition. Our finding that Cav1 is both an aggresome-inducing and aggresome-localized protein provides new insights into how cells handle and respond to misfolded Cav1. They also raise the possibility that aggresome formation may contribute to some of reported phenotypes associated with overexpressed and/or mutant forms of Cav1.
Assuntos
Caveolina 1/metabolismo , Agregados Proteicos , Animais , Células COS , Caveolina 1/genética , Chlorocebus aethiops , Imunofluorescência , Expressão Gênica , Humanos , Mecanotransdução Celular , Microtúbulos/metabolismo , Mutação , Especificidade de Órgãos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Estresse Fisiológico , UbiquitinaçãoRESUMO
The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16- an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments - is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Junções Intercelulares/metabolismo , Rim/metabolismo , Sintaxina 16/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Contagem de Células , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Colágeno , Cães , Combinação de Medicamentos , Embrião não Mamífero , Endossomos/metabolismo , Endossomos/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Junções Intercelulares/genética , Junções Intercelulares/ultraestrutura , Rim/crescimento & desenvolvimento , Rim/ultraestrutura , Laminina , Células Madin Darby de Rim Canino , Transporte Proteico , Proteoglicanas , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Sintaxina 16/genética , Transgenes , Peixe-ZebraRESUMO
Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.