Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

Abstract

We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

Cytoplasmic travels of the ecdysteroid receptor in target cells: pathways for both genomic and non-genomic actions

“…This antibody detects an epitope between residues 127 and 354 of Manduca sexta EcR; these sequences are present in all EcR isoforms of Manduca , hence they were designated EcR-common (Fujiwara et al, 1995; Jindra et al, 1996). This antibody has been used previously to localize EcR in PG cells (Vafopoulou, 2009). Several lines of evidence validated the specificity of this antibody in recognizing the EcR of Rhodnius .…”

“…Several lines of evidence validated the specificity of this antibody in recognizing the EcR of Rhodnius . First, this antibody recognizes only three proteins in Rhodnius PGs on Western blots at 79, 64, and 56 kDa (Vafopoulou, 2009). Proteins of closely similar molecular masses to those above were also identified in various Rhodnius tissue extracts using two other antibodies (15C3 and 10F1) which were also produced against the common region of Manduca EcR (Vafopoulou et al, 2005).…”

“…Membrane-bound molecules that appear to be related to conventional EcR have been reported (Elmogy et al, 2004, 2007). We reported recently that EcR associates with mitochondria in the cytoplasm of PG cells of Rhodnius (Vafopoulou, 2009). Here, we report co-localization of cytoplasmic EcR with mitochondria in all cell types studied, showing that the mitochondria are a potential site for non-genomic actions of ecdysteroids.…”

“…This shuttling rhythm persists in continuous darkness, emphasizing its circadian nature (Vafopoulou and Steel, 2006). The circadian rhythm of shuttling is in synchrony with the circadian rhythm of circulating ecdysteroids (Vafopoulou and Steel, 2006; Vafopoulou, 2009). Circadian shuttling had not been seen previously in any animal.…”

“…We proposed recently, using double immunolabels, that EcR may utilize molecular machinery involving cytoskeletal tracks, similar to vesicle transport (Vafopoulou, 2009). The cytoskeleton has been implicated as a system for targeted movement of several mammalian steroid receptors.…”