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1.
Methods Mol Biol ; 2169: 89-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548822

RESUMO

Biotin proximity labeling or BioID is a technique used to detect neighboring proteins, including transient and low-affinity interactions in their natural cellular environment. Here I describe the use of BioID in HeLa cells to identify proteins that can potentially interact with cavin1, one of the main components of caveolae. Briefly, the method consists in the transfection of the cells with the fusion constructs containing the promiscuous biotin ligase and cavin1 or control proteins, followed by biotin, cell lysis, affinity isolation of biotinylated proteins, biotin pull-down, and identification of the biotinylated proteins using mass spectrometry.


Assuntos
Biotina/química , Caveolina 1/metabolismo , Espectrometria de Massas/métodos , Proteínas de Ligação a RNA/metabolismo , Biotina/genética , Western Blotting , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Cavéolas/metabolismo , Caveolina 1/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Imunofluorescência/métodos , Células HeLa , Humanos , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transfecção
2.
Methods Mol Biol ; 2169: 149-166, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548827

RESUMO

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Caveolina 1/genética , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Transporte/metabolismo , Cavéolas/metabolismo , Caveolina 1/metabolismo , Clonagem Molecular/métodos , Eletroporação/métodos , Citometria de Fluxo , Imunofluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Plasmídeos/genética , Proteínas de Ligação a RNA/genética
3.
Curr Biol ; 27(19): 2951-2962.e5, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28943089

RESUMO

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.


Assuntos
Proteínas de Transporte/genética , Cavéolas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Fenômenos Biomecânicos , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Estresse Mecânico , Proteínas de Transporte Vesicular/metabolismo
4.
J Cell Biol ; 211(1): 53-61, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26459598

RESUMO

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1(-/-) mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo.


Assuntos
Débito Cardíaco , Cavéolas/fisiologia , Células Endoteliais/fisiologia , Animais , Fenômenos Biomecânicos , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Endocitose , Camundongos Endogâmicos C57BL , Camundongos Knockout
5.
PLoS Biol ; 11(8): e1001640, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24013648

RESUMO

Caveolae are an abundant feature of the plasma membrane of many mammalian cell types, and have key roles in mechano-transduction, metabolic regulation, and vascular permeability. Caveolin and cavin proteins, as well as EHD2 and pacsin 2, are all present in caveolae. How these proteins assemble to form a protein interaction network for caveolar morphogenesis is not known. Using in vivo crosslinking, velocity gradient centrifugation, immuno-isolation, and tandem mass spectrometry, we determine that cavins and caveolins assemble into a homogenous 80S complex, which we term the caveolar coat complex. There are no further abundant components within this complex, and the complex excludes EHD2 and pacsin 2. Cavin 1 forms trimers and interacts with caveolin 1 with a molar ratio of about 1∶4. Cavins 2 and 3 compete for binding sites within the overall coat complex, and form distinct subcomplexes with cavin 1. The core interactions between caveolin 1 and cavin 1 are independent of cavin 2, cavin 3, and EHD2 expression, and the cavins themselves can still interact in the absence of caveolin 1. Using immuno-electron microscopy as well as a recently developed protein tag for electron microscopy (MiniSOG), we demonstrate that caveolar coat complexes form a distinct coat all around the caveolar bulb. In contrast, and consistent with our biochemical data, EHD2 defines a different domain at the caveolar neck. 3D electron tomograms of the caveolar coat, labeled using cavin-MiniSOG, show that the caveolar coat is composed of repeating units of a unitary caveolar coat complex.


Assuntos
Cavéolas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte/metabolismo , Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica
6.
Proc Natl Acad Sci U S A ; 108(5): 1937-42, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21245303

RESUMO

Wnt/ß-catenin signaling controls numerous steps in normal animal development and can also cause cancer if inappropriately activated. In the absence of Wnt, ß-catenin is targeted continuously for proteasomal degradation by the Axin destruction complex, whose activity is blocked upon Wnt stimulation by Dishevelled, which recruits Axin to the plasma membrane and assembles it into a signalosome. This key event during Wnt signal transduction depends on dynamic head-to-tail polymerization by the DIX domain of Dishevelled. Here, we use rescue assays in Drosophila tissues and functional assays in human cells to show that polymerization-blocking mutations in the DIX domain of Axin disable its effector function in down-regulating Armadillo/ß-catenin and its response to Dishevelled during Wnt signaling. Intriguingly, NMR spectroscopy revealed that the purified DIX domains of the two proteins interact with each other directly through their polymerization interfaces, whereby the same residues mediate both homo- and heterotypic interactions. This result implies that Dishevelled has the potential to act as a "natural" dominant-negative, binding to the polymerization interface of Axin's DIX domain to interfere with its self-assembly, thereby blocking its effector function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biopolímeros/metabolismo , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Fosfoproteínas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Proteína Axina , Proteínas Desgrenhadas , Drosophila , Proteínas de Drosophila/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Ligação Proteica , Homologia de Sequência de Aminoácidos
7.
Open Biol ; 1(3): 110013, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22645652

RESUMO

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector ß-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, we discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Axina/metabolismo , Complexo de Sinalização da Axina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Animais , Animais Geneticamente Modificados , Proteína Axina/química , Proteína Axina/genética , Complexo de Sinalização da Axina/química , Complexo de Sinalização da Axina/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas Desgrenhadas , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Endorribonucleases/metabolismo , Genes APC , Genes de Insetos , Humanos , Complexos Multienzimáticos/metabolismo , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/metabolismo , Via de Sinalização Wnt
8.
J Cell Sci ; 123(Pt 9): 1588-99, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20388731

RESUMO

Wnt/beta-catenin signalling controls cell fates in development, tissue homeostasis and cancer. Wnt binding to Frizzled receptors triggers recruitment of Dishevelled to the plasma membrane and formation of a signalosome containing the LRP5/6 co-receptor, whose cytoplasmic tail (ctail) thus becomes phosphorylated at multiple PPP(S/T)Px(S/T) motifs. These then directly inhibit GSK3beta, which results in beta-catenin accumulation and signalling. Here, we revisit previous epistasis experiments, and show that Dishevelled signals through LRP5/6 in human cells and Drosophila embryos. To recapitulate this signalling event, and to define its functional elements, we fused the Dishevelled DIX domain to the LRP6 ctail, which forms cytoplasmic signalosomes with potent signalling activity mediated by its PPP(S/T)Px(S/T) motifs. Their phosphorylation and activity depends critically on DIX-mediated polymerization, and on multiple stability elements in the LRP6 ctail, including the T1479 epitope upstream of the membrane-proximal PPP(S/T)Px(S/T) motif. Thus, stable polymerization emerges as a key principle underlying the function of Dishevelled-dependent signalosomes.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Biopolímeros/metabolismo , Drosophila melanogaster/metabolismo , Fosfoproteínas/química , Estabilidade Proteica , Receptores de LDL/química , Receptores de LDL/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteína Axina , Linhagem Celular , Proteínas Desgrenhadas , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Epitopos/química , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Proteínas Wnt/metabolismo
9.
Gene Expr Patterns ; 8(6): 443-451, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18501681

RESUMO

The products of the Drosophila discs-large (dlg) gene are members of the MAGUK family of proteins, a group of proteins involved in localization, transport and recycling of receptors and channels in cell junctions, including the synapse. In vertebrates, four genes with multiple splice variants homologous to dlg are described. dlg originates two main proteins, DLGA, similar to the vertebrate neuronal protein PSD95, and DLGS97, similar to the vertebrate neuronal and epithelial protein SAP97. DLGA is expressed in epithelia, neural tissue and muscle. DLGS97 is expressed in neural tissue and muscle but not in epithelia. The distinctive difference between them is the presence in DLGS97 of an L27 domain. The differential expression between these variants makes the study of DLGS97 of key relevance to understand the in vivo role of synaptic MAGUKs in neurons. Here we present the temporal and spatial expression pattern of DLGS97 during embryonic and larval nervous system development, during eye development and in adult brain. Our results show that DLGS97 is expressed zygotically, in neurons in the embryo, larvae and adult, and is absent at all stages in glial cells. During eye development DLGS97 starts to be expressed in photoreceptor cells at early stages of differentiation and localizes basal to the basolateral junctions. In the brain, DLGS97 is expressed in the mushroom bodies and optic lobes at larval and adult stages; and in the antennal lobe in the adult stage. In addition we show that both, dlgS97 and dlgA transcripts, express during development multiple splice variants with differences in the use of exons in two sites.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Processamento Alternativo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Drosophila/embriologia , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras de Invertebrados/embriologia , Células Fotorreceptoras de Invertebrados/metabolismo , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
10.
J Neurosci ; 28(1): 304-14, 2008 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18171947

RESUMO

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses.


Assuntos
Comportamento Animal/fisiologia , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Junção Neuromuscular/fisiologia , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Animais Geneticamente Modificados , Ritmo Circadiano/fisiologia , Drosophila , Proteínas de Drosophila/genética , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/fisiologia , Microscopia Eletrônica de Transmissão/métodos , Atividade Motora , Mutação/fisiologia , Junção Neuromuscular/ultraestrutura , Isoformas de Proteínas/genética , Comportamento Sexual Animal/fisiologia , Proteínas Supressoras de Tumor/genética
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