RESUMEN
The original concept that lipid and protein components are randomly distributed in cellular membranes has been challenged by evidence of compartmentalization of such components into discrete membrane microdomains (known as lipid rafts). The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry to test the idea that such domains concentrate machinery to mediate cellular processes such as signaling, synaptic plasticity, and endocytosis. As such, a large number of studies have used biochemical, cell biological, and biophysical methodologies to define the composition of membrane microdomains in experimental contexts. Although biochemical preparation strategies are not without limitations (as discussed herein), the isolation of detergent-resistant and detergent-free membrane domains can provide important information about the segregation of lipids and proteins in membranes. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains and also provide methods for subsequent classical or mass spectrometry-based lipid analytical approaches.
Asunto(s)
Ácidos Grasos , Microdominios de Membrana , Ácidos Grasos/metabolismo , Microdominios de Membrana/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Detergentes/químicaRESUMEN
Purpose: The immune-privileged environment and complex organization of retinal tissue support the retina's essential role in visual function, yet confound inquiries into cell-specific inflammatory effects that lead to dysfunction and degeneration. Caveolin-1 (Cav1) is an integral membrane protein expressed in several retinal cell types and is implicated in immune regulation. However, whether Cav1 promotes or inhibits inflammatory processes in the retina (as well as in other tissues) remains unclear. Previously, we showed that global-Cav1 depletion resulted in reduced retinal inflammatory cytokine production but paradoxically elevated retinal immune cell infiltration. We hypothesized that these disparate responses are the result of differential cell-specific Cav1 functions in the retina. Methods: We used Cre/lox technology to deplete Cav1 specifically in the neural retinal (NR) compartment to clarify the role NR-specific Cav1 (NR-Cav1) in the retinal immune response to intravitreal inflammatory challenge induced by activation of Toll-like receptor-4 (TLR4). We used multiplex protein suspension array and flow cytometry to evaluate innate immune activation. Additionally, we used bioinformatics assessment of differentially expressed membrane-associated proteins to infer relationships between NR-Cav1 and immune response pathways. Results: NR-Cav1 depletion, which primarily affects Müller glia Cav1 expression, significantly altered immune response pathway regulators, decreased retinal inflammatory cytokine production, and reduced retinal immune cell infiltration in response to LPS-stimulated inflammatory induction. Conclusions: Cav1 expression in the NR compartment promotes the innate TLR4-mediated retinal tissue immune response. Additionally, we have identified novel potential immune modulators differentially expressed with NR-Cav1 depletion. This study further clarifies the role of NR-Cav1 in retinal inflammation.
Asunto(s)
Caveolina 1/fisiología , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Retina/metabolismo , Retinitis/inducido químicamente , Animales , Western Blotting , Caveolina 1/deficiencia , Citocinas/metabolismo , Sinergismo Farmacológico , Electrorretinografía , Citometría de Flujo , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/patología , Inyecciones Intravítreas , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Nistagmo Optoquinético/fisiología , Proteómica , Retinitis/metabolismo , Retinitis/patología , Salmonella typhimurium , Receptor Toll-Like 4/metabolismoRESUMEN
Purpose: Polymorphisms at the caveolin-1/2 locus are associated with glaucoma and IOP risk and deletion of caveolin-1 (Cav1) in mice elevates IOP and reduces outflow facility. However, the specific location/cell type responsible for Cav1-dependent regulation of IOP is unclear. We hypothesized that endothelial Cav1 in the conventional outflow (CO) pathway regulate IOP via endothelial nitric oxide synthase (eNOS) signaling. Methods: We created a mouse with targeted deletion of Cav1 in endothelial cells (Cav1ΔEC) and evaluated IOP, outflow facility, outflow pathway distal vascular morphology, eNOS phosphorylation, and tyrosine nitration of iridocorneal angle tissues by Western blotting. Results: Endothelial deletion of Cav1 resulted in significantly elevated IOP versus wild-type mice but not a concomitant decrease in outflow facility. Endothelial Cav1 deficiency did not alter the trabecular meshwork or Schlemm's canal morphology, suggesting that the effects observed were not due to developmental deformities. Endothelial Cav1 deletion resulted in eNOS hyperactivity, modestly increased protein nitration, and significant enlargement of the drainage vessels distal to Schlemm's canal. L-Nitro-arginine methyl ester treatment reduced outflow in Cav1ΔEC but not wild-type mice and had no effect on the size of drainage vessels. Endothelin-1 treatment decrease the outflow and drainage vessel size in both wild-type and Cav1ΔEC mice. Conclusions: Our results suggest that hyperactive eNOS signaling in the CO pathway of both Cav1ΔEC and global Cav1 knockout mice results in chronic dilation of distal CO vessels and protein nitration, but that Cav1 expression in the trabecular meshwork is sufficient to rescue CO defects reported in global Cav1 knockout mice.
Asunto(s)
Humor Acuoso/metabolismo , Caveolina 1/genética , ADN/genética , Células Endoteliales/metabolismo , Glaucoma/genética , Presión Intraocular/fisiología , Polimorfismo Genético , Animales , Western Blotting , Caveolina 1/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/patología , Glaucoma/metabolismo , Glaucoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
The compartmentalization of cellular membranes into discrete membrane microdomains (known as lipid rafts) challenged the original definition of membranes as containing randomly distributed lipid and protein components. The lipid microdomain hypothesis has generated significant controversy and rigorous inquiry based on the attractive idea that such domains concentrate machinery to mediate cellular events such as signaling and endocytosis. As such, numerous studies have used biochemical, cell biological, and biophysical methodologies to define the composition of such domains in a variety of experimental contexts. In this chapter, we describe methodologies to isolate membranes from cell or tissue sources with biophysical/biochemical properties of membrane microdomains that are amenable to subsequent classical or mass spectrometry-based lipid analytical approaches.
Asunto(s)
Colesterol/química , Detergentes/química , Ácidos Grasos/química , Microdominios de Membrana , Carbonatos/química , Centrifugación por Gradiente de Densidad , Colesterol/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Ácidos Grasos/aislamiento & purificación , Lípidos/química , Lípidos/aislamiento & purificaciónRESUMEN
Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function.
Asunto(s)
Barrera Hematorretinal , Caveolas/metabolismo , Caveolinas/metabolismo , Glaucoma/metabolismo , Presión Intraocular/fisiología , Mecanotransducción Celular/fisiología , Fenómenos Fisiológicos Oculares , Animales , Glaucoma/fisiopatología , HumanosRESUMEN
Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1-/-) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1-/- outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1-/- eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.
Asunto(s)
Caveolas/metabolismo , Caveolina 1/metabolismo , Glaucoma/metabolismo , Presión Intraocular , Malla Trabecular/metabolismo , Animales , Caveolas/patología , Caveolina 1/genética , Glaucoma/genética , Glaucoma/patología , Glaucoma/fisiopatología , Humanos , Ratones , Ratones Noqueados , Malla Trabecular/patología , Malla Trabecular/fisiopatologíaRESUMEN
Caveolin-1 (Cav-1), an integral component of caveolar membrane domains, is expressed in several retinal cell types, including photoreceptors, retinal vascular endothelial cells, Müller glia, and retinal pigment epithelium (RPE) cells. Recent evidence links Cav-1 to ocular diseases, including autoimmune uveitis, diabetic retinopathy, and primary open angle glaucoma, but its role in normal vision is largely undetermined. In this report, we show that ablation of Cav-1 results in reduced inner and outer retinal function as measured, in vivo, by electroretinography and manganese-enhanced MRI. Somewhat surprisingly, dark current and light sensitivity were normal in individual rods (recorded with suction electrode methods) from Cav-1 knock-out (KO) mice. Although photoreceptor function was largely normal, in vitro, the apparent K(+) affinity of the RPE-expressed α1-Na(+)/K(+)-ATPase was decreased in Cav-1 KO mice. Cav-1 KO retinas also displayed unusually tight adhesion with the RPE, which could be resolved by brief treatment with hyperosmotic medium, suggesting alterations in outer retinal fluid homeostasis. Collectively, these findings demonstrate that reduced retinal function resulting from Cav-1 ablation is not photoreceptor-intrinsic but rather involves impaired subretinal and/or RPE ion/fluid homeostasis.
Asunto(s)
Caveolina 1/metabolismo , Microambiente Celular/fisiología , Homeostasis/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Uniones Estrechas/metabolismo , Animales , Caveolina 1/genética , Ratones , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/citología , Potasio/metabolismo , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , ATPasa Intercambiadora de Sodio-Potasio/genética , Uniones Estrechas/genéticaRESUMEN
PURPOSE: Recent evidence suggests that ceramide metabolism plays an important role in retinal photoreceptor cell survival and apoptosis. The purpose of this study was to characterize sphingolipids in the retina with special emphasis on the very-long-chain-containing saturated (VLC-FA) and polyunsaturated (VLC-PUFA) fatty acid-containing species. The VLC-FAs and VLC-PUFAs are synthesized by the ELOVL4 protein, which is involved in human Stargardt's macular dystrophy type 3 (STGD3). METHODS: Total lipids were extracted from retina and other tissues, and different sphingolipid classes were isolated and purified using various combinations of liquid- and solid-phase separation. Purified sphingolipids were analyzed by high-performance thin layer chromatography (HPTLC), gas chromatography (GC), and GC-MS (GC-mass spectrometry). RESULTS: Nonsialylated sphingolipids (NSLs) comprised approximately 3.5% of total retinal lipids of which 70% was sphingomyelin. Ceramide and glycosylceramides (GCs) constitutedAsunto(s)
Ácidos Grasos Insaturados/metabolismo
, Ácidos Grasos/metabolismo
, Retina/metabolismo
, Esfingolípidos/metabolismo
, Animales
, Encéfalo/metabolismo
, Bovinos
, Membrana Celular/metabolismo
, Cromatografía de Gases
, Cromatografía en Capa Delgada
, Ácidos Grasos/aislamiento & purificación
, Ácidos Grasos Insaturados/aislamiento & purificación
, Cromatografía de Gases y Espectrometría de Masas
, Hígado/metabolismo
, Masculino
, Especificidad de Órganos
, Ratas
, Especies Reactivas de Oxígeno/metabolismo
, Piel/metabolismo
, Esfingolípidos/aislamiento & purificación
, Testículo/metabolismo
RESUMEN
Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.
Asunto(s)
Colesterol/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Lípidos de la Membrana/química , Microdominios de Membrana/efectos de los fármacos , Esfingolípidos/química , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ácidos Grasos Monoinsaturados/farmacología , Humanos , Riñón/embriología , Microdominios de Membrana/metabolismo , Retina/ultraestructura , Esfingolípidos/antagonistas & inhibidores , Esfingolípidos/biosíntesis , beta-Ciclodextrinas/farmacologíaRESUMEN
Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.
Asunto(s)
Membrana Celular/química , Membrana Celular/efectos de los fármacos , Detergentes/farmacología , Lípidos , Proteínas/metabolismo , Segmento Externo de la Célula en Bastón/ultraestructura , Animales , Bovinos , Microscopía Inmunoelectrónica/métodos , Proteínas/efectos de los fármacos , Proteómica/métodos , Retina/citología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosAsunto(s)
Detergentes/farmacología , Microdominios de Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/biosíntesis , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor de Insulina/biosíntesis , Segmento Externo de la Célula en Bastón/metabolismo , Animales , Western Blotting , Bovinos , Electroforesis en Gel de Poliacrilamida , Lípidos/química , Microdominios de Membrana/enzimología , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Tirosina/química , beta-Ciclodextrinas/químicaRESUMEN
Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Receptor de Insulina/metabolismo , Retina/enzimología , Retina/metabolismo , Células 3T3-L1 , Secuencia de Aminoácidos , Animales , Insulina/química , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/química , Receptor de Insulina/genética , Técnicas del Sistema de Dos Híbridos , Tirosina/genética , Tirosina/metabolismoAsunto(s)
Fosfatidilinositol 3-Quinasas/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Retina/enzimología , Animales , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor de Insulina/metabolismo , Retina/metabolismo , Retina/patologíaRESUMEN
Recently, we have shown that phosphoinositide 3-kinase (PI3K) in bovine rod outer segment (ROS) is activated in vitro by tyrosine phosphorylation of the C-terminal tail of the insulin receptor (Rajala, R. V. S., and Anderson, R. E. (2001) Invest. Ophthal. Vis. Sci. 42, 3110-3117). In this study, we have investigated the in vivo mechanism of PI3K activation in the rodent retina and report the novel finding that light stimulates tyrosine phosphorylation of the beta-subunit of the insulin receptor (IRbeta) in ROS membranes, which leads to the association of PI3K enzyme activity with IRbeta. Retinas from light- or dark-adapted mice and rats were homogenized and immunoprecipitated with antibodies against phosphotyrosine, IRbeta, or the p85 regulatory subunit of PI3K, and PI3K activity was measured using PI-4,5-P(2) as substrate. We observed a light-dependent increase in tyrosine phosphorylation of IRbeta and an increase in PI3K enzyme activity in isolated ROS and in anti-phosphotyrosine and anti-IRbeta immunoprecipitates of retinal homogenates. The light effect was localized to photoreceptor neurons and is independent of insulin secretion. Our results suggest that light induces tyrosine phosphorylation of IRbeta in outer segment membranes, which leads to the binding of p85 through its N-terminal Src homology 2 domain and the generation of PI-3,4,5-P(3). We suggest that the physiological role of this process may be to provide neuroprotection of the retina against light damage by activating proteins that protect against stress-induced apoptosis.