RESUMO
Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ≈ 20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.
Assuntos
Retículo Endoplasmático/química , Macrófagos/metabolismo , Fagossomos/química , Proteômica/métodos , Animais , Biomarcadores/análise , Western Blotting , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/química , Endossomos/metabolismo , Endossomos/ultraestrutura , Imunofluorescência , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Marcação por Isótopo , Lisossomos/química , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Macrófagos/citologia , Macrófagos/ultraestrutura , Espectrometria de Massas , Camundongos , Oligopeptídeos , Fagocitose , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Plasmídeos , Sinais Direcionadores de Proteínas , Proteínas Qa-SNARE , TransfecçãoRESUMO
Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1(Δ317-333)) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1(WT) mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1(Δ317-333), suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.
Assuntos
Proteínas Munc18/química , Proteínas Munc18/metabolismo , Nanopartículas/química , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo , Animais , Área Sob a Curva , Toxinas Botulínicas/metabolismo , Humanos , Modelos Moleculares , Células PC12 , Domínios Proteicos , Estrutura Secundária de Proteína , RatosRESUMO
Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Degeneração Neural , Neurônios/metabolismo , Agregados Proteicos , alfa-Sinucleína/metabolismo , Animais , Animais Recém-Nascidos , Genótipo , Haploinsuficiência , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Microscopia de Fluorescência , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Proteínas Munc18/química , Proteínas Munc18/genética , Mutação , Neurônios/patologia , Células PC12 , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ligação Proteica , Conformação Proteica , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.
Assuntos
Endocitose , Movimento (Física) , Fenômenos Ópticos , Vesículas Sinápticas/metabolismo , Animais , Axônios/metabolismo , Teorema de Bayes , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Imageamento Tridimensional , Cadeias de Markov , Neurônios/metabolismo , Ratos Sprague-Dawley , Anticorpos de Domínio Único/metabolismo , Processos Estocásticos , Sinapses/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
We have prepared a number of silicone-based thermoplastic polyurethane (TPU) nanocomposites and demonstrated an enhancement of in vitro biostability against metal-ion-induced oxidation for potential use in long-term implantable medical devices. Organoclays based on both low-aspect-ratio hectorites and high-aspect-ratio fluoromicas were evaluated after being dual-modified with two quaternary alkyl ammonium salts with differing degrees of polarity. The resultant nanocomposites were tested for in vitro biostability using physiologically relevant oxidizing conditions. Subsequently, the effects of oxidative treatment on the surface degradation and bulk mechanical integrity of the nanocomposites were investigated and compared with the parent TPUs to identify nanocomposites with the most desirable features for long-term implantation. Here, we demonstrate that the low-aspect-ratio organohectorite was delaminated and well dispersed in the nanocomposites. Importantly, these factors gave rise to the enhanced oxidative stability. In addition, the mechanical properties of all nanocomposites were less adversely affected by the oxidative treatment compared to their parent TPUs. These results suggest the potential for improved mechanical integrity and biostability when suitable dual modified organoclays are incorporated in a silicone-based TPU.
Assuntos
Materiais Biocompatíveis/química , Dimetilpolisiloxanos/química , Metais/química , Nanoestruturas/química , Poliuretanos/química , Silicatos/química , Silicatos de Alumínio/química , Argila , Estabilidade de Medicamentos , Teste de Materiais , Nanoestruturas/ultraestrutura , Tamanho da PartículaRESUMO
Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.
Assuntos
Cálcio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Vesículas Secretórias/metabolismo , Androstadienos/metabolismo , Animais , Bovinos , Linhagem Celular , Células Cromafins/citologia , Células Cromafins/metabolismo , Classe II de Fosfatidilinositol 3-Quinases , Exocitose/fisiologia , Humanos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , WortmaninaRESUMO
While our understanding of lipid microdomains has advanced in recent years, many aspects of their formation and dynamics are still unclear. In particular, the molecular determinants that facilitate the partitioning of integral membrane proteins into lipid raft domains are yet to be clarified. This review focuses on a family of raft-associated integral membrane proteins, termed flotillins, which belongs to a larger class of integral membrane proteins that carry an evolutionarily conserved domain called the prohibitin homology (PHB) domain. A number of studies now suggest that eucaryotic proteins carrying this domain have affinity for lipid raft domains. The PHB domain is carried by a diverse array of proteins including stomatin, podocin, the archetypal PHB protein, prohibitin, lower eucaryotic proteins such as the Dictyostelium discoideum proteins vacuolin A and vacuolin B and the Caenorhabditis elegans proteins unc-1, unc-24 and mec-2. The presence of this domain in some procaryotic proteins suggests that the PHB domain may constitute a primordial lipid recognition motif. Recent work has provided new insights into the trafficking and targeting of flotillin and other PHB domain proteins. While the function of this large family of proteins remains unclear, studies of the C. elegans PHB proteins suggest possible links to a class of volatile anaesthetics raising the possibility that these lipophilic agents could influence lipid raft domains. This review will discuss recent insights into the cell biology of flotillins and the large family of evolutionarily conserved PHB domain proteins.
Assuntos
Anestésicos/química , Anestésicos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proibitinas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.
Assuntos
Proteínas de Peixes , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ácido Palmítico/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Brefeldina A/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Citoplasma/metabolismo , Primers do DNA , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Transporte Proteico , Homologia de Sequência de Aminoácidos , Células VeroRESUMO
Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.
Assuntos
Caveolinas/genética , Caveolinas/farmacologia , Colesterol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Distrofias Musculares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Caveolina 3 , Linhagem Celular , Cricetinae , Genes ras , Complexo de Golgi/metabolismo , Músculos/metabolismo , Distrofias Musculares/genética , Células PC12 , Mutação Puntual , RatosRESUMO
The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.