Overexpression of kermit/dGIPC is associated with lethality in Drosophila melanogaster

Insertional mutagenesis is an important tool for functional genomics in Drosophila melanogaster. The insertion site in the KG00562 mutant fly line has been mapped to the CG8709 (herein named DmLpin) locus and to the 3’ of kermit (also called dGIPC). This mutant line presents a high lethality rate resulting from a gain of function. To obtain some insight into the biological role of the mutated locus, we have characterized the mutation and its relation to the high mortality of the KG00562 fly line. In this mutant, we did not detect one of the DmLpin transcripts, namely DmLpinK, but we did detect an unusual 2.3-kb mRNA (LpinK-w). Further investigation revealed that the LpinK-w transcript results from an aberrant splicing between the untranslated first exon of DmLpinK and the mini-white marker gene. Lack of DmLpinK or LpinK-w expression does not contribute to lethality, since heterozygous KG00562/Def7860 animals presented lethality rates comparable to those of the wild type. In contrast, the overexpression of kermit was associated with lethality of the KG00562 fly line. Significantly higher levels of kermit were detected in the Malpighian tubules of KG00562/+ flies that presented higher lethality rates than wild-type or KG00562/Def7860 animals, in which the lethality was rescued. In agreement with a recently reported study, our data support the hypothesis that misexpression of kermit/dGIPC could interfere with Drosophila development, with further investigations being needed in this direction.

Estudio de los mecanismos de transporte iónico involucrados en la secreción en túbulos de malpighi de chipo (Rhodnius prolixus) / Study of the mechanism of ionic transport involved on the secretion of malpighian tubes of chipos (Rhodnius prolixus)

Se midió la tasa de secreción y los cambios en la concentración interna de sodio y protones de los túbulos demalpighi (UMT) en Rhodnius prolixus bajo estimulación con 5-OH-Triptamina. Para medir secreción utilizamos un sistema de doble perfusión para tener acceso por separado a las membranas basolateral y/o apical de la célula. Los flujos iónicos se midieron por microfluorometría. Se aplicaron hasta trece agentes farmacológicos: Ouabaina, Bafilomicina A1, Furosamida, Bumetanida, DIOA, Probenecina, SITS, Acetazolamida, Amilorida, DPC, BaCl2, pCMBS y DTT. Estos agentes son bloqueadores conocidos de diferentes funciones de transporte de iones, conocidos como ATPasas, co- y/o contratransportadores asi como de canales iónicos y de agua. La asunción básica es que los cambios en los movimientos de agua reflejan los cambios en los mecanismos del transporte de iones que se localizan de la siguiente manera: (i) en la membrana basolateral de la célula, los principales son un cotransportador de Na+-K+-2Cl– y el intercambiador de Cl--HCO- 3; la ATPasa de Na+- K+ y la ATPasa de Na+ y los canales de Cl- y de agua (Rp-MIP) son de importancia intermedia, mientras que los canales de K+ son menos importantes: (ii) en la membrana celular apical, la mayor importancia la tiene un cotransportador de K+-Cl-, que ha sido localizado por primera vez, una ATPasa de H+ tipo V, y un intercambiador de Na+-H+; el intercambiador urato-anión y los canales de K+ son importantes mientras que los canales de Clno lo son. En este trabajo presentamos un modelo actualizado que explica la secreción del UMT.

We have measured fluid secretion rate and intracelular concentration of sodium and protons, in Rhodnius prolixusUMT stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. The ionic fluxes has been measured using microfluorimetry. 13 pharmacological agents were applied: Ouabain, Bafilomycin A1, Furosemide, Bumetanide, DIOA, Probenecid, SITS, Acetazolamide, Amiloride, DPC, BaCl2, pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter- transporters and ionic and water channels. The basic assumption is that water movement changes reflect changes on ion transport mechanisms which we localize as follows: (i) At the basolateral cell membrane: a Na+ - K+ - 2Cl- cotransport; a Cl- - HCO3 - exchange; the Na+ - K+ - ATPase, Cl- channels and Rp-MIP water channels are fundamental; K+ channels play a lesser role. (ii) At the apical cell membrane, a K+ - Cl- cotransport that is located for the first time; a V - H+ - ATPase; an Na+– H+ exchange; an urate - anion exchange and K+ channels are important, while Cl- channels are not. The present experiments allow us to build a tentative model for the function of the UMT cell, which includes a paracellular pathway for fluid flow. In this work we propose a new actualized model to explain the UMT secretion.