RESUMEN
Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.
Asunto(s)
Activación del Canal Iónico/efectos de la radiación , Luz , Microvellosidades/metabolismo , Microvellosidades/efectos de la radiación , Células Fotorreceptoras de Invertebrados/citología , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/efectos de la radiación , Animales , Diacilglicerol Quinasa/biosíntesis , Diglicéridos/farmacología , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efectos de la radiación , Drosophila melanogaster , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Potenciales de la Membrana/efectos de los fármacos , Proteína Quinasa C/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sulfonamidas/farmacología , Canales de Potencial de Receptor Transitorio/aislamiento & purificación , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/genéticaRESUMEN
Coimmunoprecipitation (coIP) provides evidence that two or more proteins can be found in the same complex. It can be performed in vitro (employing in vitro transcribed and translated proteins, or proteins expressed in Escherichia coli) or from transfected cells, which assess whether the interaction takes place in a more functional context. This chapter includes a general description and guidelines to carry out coIP in transfected Schneider's cells.
Asunto(s)
Proteínas de Drosophila/aislamiento & purificación , Drosophila/química , Inmunoprecipitación/métodos , Animales , Línea Celular , Drosophila/citología , Electroforesis en Gel de Poliacrilamida , Lípidos , Proteínas Recombinantes/aislamiento & purificación , TransfecciónRESUMEN
The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.
Asunto(s)
Culicidae/genética , Proteínas de Drosophila/aislamiento & purificación , Drosophila/genética , Insectos Vectores/genética , Proteínas Ribosómicas/aislamiento & purificación , Aedes/genética , Animales , Anopheles/genética , Culex/genética , Cartilla de ADN/genética , ADN Complementario/genética , Proteínas de Drosophila/genética , Expresión Génica , Reacción en Cadena de la Polimerasa , Proteínas Ribosómicas/genéticaRESUMEN
The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.