Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster.Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. Here, we present evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the winstrol depot orally caused developmental defects due to deficiency of vesicle-mediated steroid hormone secretion in drosophila melanogaster. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. We propose that trafficking of steroid hormones vesicle-mediated steroid hormone secretion in drosophila melanogaster of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process. National Center for Biotechnology InformationU.
PlumX - Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster.
Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues.
Here we present evidence, that in Drosophila , endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism.
Inhibition of calcium signaling in the steroidogenic prothoracic gland results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone.
Accumulation of Synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. We propose that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process.
Steroid hormones are an important class of bioactive molecules in both animal and plant kingdoms that regulate a wide variety of physiological processes including immune response, salt and water balance, glucose metabolism, and sexual maturation during juvenile development Sapolsky et al.
In insect larvae, the primary precursor steroid hormone ecdysone E is produced in the prothoracic gland PG. After its release into the circulatory system, E is taken up in peripheral tissues such as the gut and fat body where it is converted to hydroxyecdysone 20E.
This is the active derivative that regulates larval molt timing and the onset of metamorphosis leading to the formation of sexually mature adults Yamanaka et al.
The biosynthetic pathways of steroid hormone production have been extensively studied in diverse animal species, and many of the key enzymes have been identified and well characterized Ghayee and Auchus, ; Huang et al. In insects, E biosynthesis is stimulated by extracellular signaling molecules such as the prothoracicotropic hormone PTTH , which binds to its receptor Torso to induce the expression of genes encoding steroidogenic enzymes Rewitz et al.
In contrast to the extensive literature describing studies on steroidogenic processes, very little is known about the mechanisms that regulate release of steroid hormones from endocrine tissues. Indeed, the textbook view is that lipophilic steroid hormones simply enter and exit cells by diffusion across lipid bilayers Raven and Johnson, ; Sherwood, ; White and Porterfield, However, this prevailing assumption has not been extensively tested in vivo , and the limited studies described so far primarily used in vitro or in silico approaches Oren et al.
Given the scarcity of knowledge concerning this fundamental aspect of endocrinology, we used molecular genetic tools to investigate the mechanism of E release from the PG in Drosophila melanogaster. We found that blocking calcium signaling through RNAi-mediated knockdown of the inositol 1,4,5,-trisphosphate receptor IP3R in the PG leads to a buildup of E and a decrease of 20E in source and target tissues, respectively. This results in severe delay or larval developmental arrest that can be rescued by feeding larvae E.
Identical developmental defects were observed in larvae in which cellular components normally involved in calcium-mediated vesicle exocytosis, such as Rab3, UNC or Synaptotagmin 1 Syt1 , were depleted in the PG. Moreover, GCaMP imagining of the PG just prior to metamorphosis revealed spontaneous calcium signaling that was attenuated by RNAi-mediated knockdown of the upstream signaling component that couples G protein-coupled receptors GPCRs to calcium release.
Furthermore, the accumulation of Syt1-positive vesicles was observed when calcium signaling was blocked in the PG, suggesting that calcium-mediated vesicle exocytosis is required for E release.
Consistent with this notion, we identified an ABC transporter found in these Syt1-positive vesicles and show that it transports E across a lipid bilayer in vitro. Taken together, these results support a new hypothesis that transport of steroid hormones across lipid bilayers can involve a regulated vesicle-release process instead of, or in addition to, passive or facilitated diffusion mechanisms.
Studies using isolated PGs of lepidopteran insect species have long suggested a key role for calcium in stimulating E production and release in response to PTTH Huang et al. To test this in Drosophila , we conducted PG-specific knockdown of IP3R , which encodes an intracellular calcium-release channel. IP3R is highly expressed in the PG and mutants have growth defects due to low systemic levels of E Venkatesh and Hasan, , but direct links between the IP3R function and E production or release have yet to be tested.
Those larvae that arrested in the third instar stage showed an overgrowth phenotype due to an extended larval feeding period, which is commonly observed for E-deficient animals Figure 1B Caceres et al.
Feeding E to these larvae rescued the arrest phenotype, further suggesting that the systemic E level is low in these animals. Importantly, however, neither the larval arrest nor pupariation delay phenotype was fully rescued by overexpressing Ras V12 , the active form of Ras that is able to completely rescue a PTTH signaling deficiency Figures 1A and 1C Rewitz et al.
Such partial rescue strongly suggests that, unlike previous assumptions based on moth studies Huang et al. One hypothesis we considered was that calcium signaling might play a role in E release from the PG as opposed to, or in addition to, E synthesis. The small size of the RG presents a substantial dissection challenge. This analysis revealed that the titers of both E and 20E increase in the CNS-RG complex of control larvae during the wandering stage as they prepare for metamorphosis Figure 1F.
This observation is consistent with the notion that the E biosynthetic activity of the PG is stimulated by PTTH during this period and the increased levels of 20E in the hemolymph help initiate wandering behavior when taken up by the brain Yamanaka et al.
In control larvae, the titer of 20E was substantially higher than that of E in the CNS-RG complex during the wandering stage, indicating that the release of E into the hemolymph and its conversion to 20E happens quickly under normal conditions. Indeed, the 20E titer in the hemolymph increased rapidly in wandering control larvae, while the E titer remained constant or decreased during this stage Figure 1G.
For IP3R -knockdown animals that are developmentally delayed, we used wandering as a behavioral trait and used h after egg laying AEL wandering larvae for hormone titer measurement, in order to ensure that we are examining hormone titers at the same developmental stage. Consistent with this notion, the hemolymph levels of ecdysteroids in these IP3R -knockdown animals were comparable to those of early wandering control animals Figure 1G.
From these results, we infer that: The above results led us to hypothesize that E secretion from the PG is not a fully passive or facilitated diffusion process, but instead may employ a regulatory mechanism that is under the control of calcium signaling.
This is reminiscent of secretory vesicle exocytosis in neurons and endocrine cells, where the fusion of vesicles containing neurotransmitters, or various types of hormones, with the plasma membrane is tightly regulated by multiple components that sense intracellular calcium concentrations Rizo and Rosenmund, ; Sudhof, Therefore, we conducted RNAi knockdown screening of components known to be either upstream regulators of intracellular calcium release or downstream effectors of calcium-regulated secretory vesicle exocytosis Table 1 and Figure 2A.
There are two distinct genes encoding intracellular calcium-release channels in Drosophila: The knockdown of IP3R in the PG showed the larval arrest phenotype as described above, whereas that of RyR did not cause any discernible defect Table 1.
Indeed, the PG-specific knockdown of Syb , one of the two synaptobrevins in Drosophila , causes the larval arrest phenotype, supporting the hypothesis that calcium-regulated vesicle exocytosis is required for E secretion Table 1. The SNARE complex, however, is involved in all intracellular membrane fusion events, and each organism has multiple SNARE proteins that are localized to distinct membrane compartments to specify intracellular compartmental identity Li and Chin, These diverse SNARE complex functions make it difficult to interpret the above result, since E synthesis in the PG involves the trafficking of synthetic intermediates between organelles, and the disruption of this process is expected to cause similar developmental defects.
Likewise, the necessity of the exocyst complex components in the PG Table 1 and Andrews et al. Such a defect will likely disrupt several signaling pathways including PTTH and insulin signaling, both of which require the transport of their receptors to the PG plasma membrane for high level E production Yamanaka et al. In light of these difficulties in interpreting the phenotypes produced by knockdown of general secretory machinery subunits, we focused our analysis on components that are more specifically involved in calcium-regulated exocytosis.
UNC is a highly conserved, plasma membrane-associated presynaptic protein with calcium-binding domains. It interacts with the SNARE protein syntaxin and primes synaptic vesicles for fusion, and is essential for calcium-regulated synaptic vesicle exocytosis Aravamudan et al. Synaptotagmins, which form another class of calcium sensor proteins critical for vesicle exocytosis Chapman, ; de Wit et al.
The knockdown phenotypes of these regulatory exocytosis components were strikingly similar to that of IP3R ; they all showed polyphasic larval developmental arrest and pupariation delay accompanied by overgrowth, both of which were rescued by E feeding Figure 2.
To further validate that the calcium signaling pathway is active in the PG cells at the time of metamorphosis initiation, we monitored calcium dynamics in these cells using the genetically encoded calcium indicator, GCaMP5 Akerboom et al. As illustrated in Figures 2D and 2E , two types of spontaneous activities were observed in the PG cells of wandering larvae. One consisted of major concentration changes throughout the entire volume of a PG cell, which we refer to as macro spikes Figures 2D and 2E and Movie S1.
The number of active cells within a gland varied significantly, and the dynamics of the calcium concentration observed also varied in amplitude, duration and frequency on a cell by cell basis Figure 2E. A second activity, which we refer to as micro spikes Figure 2E , appeared to occur in a limited area on the cell surface and exhibited faster kinetics.
When the PG-specific knockdown of Plc21C was performed with two distinct RNAi constructs, a significant decrease in the number of animals exhibiting calcium dynamics of either class was observed Figure 2F , while RNAi of a random control gene gbb had no effect.
These results support the notion that GPCR-mediated calcium signaling is occurring in the PG cells prior to metamorphosis. In order to visualize putative secretory vesicles whose exocytosis is regulated by calcium signaling we expressed in the PG eGFP-tagged Syt1 Syt-GFP , a widely used secretory vesicle marker in both neuronal and non-neuronal cells Sugita et al. IP3R knockdown in the PG did not alter the gross morphology of the PG Figures 3B and S2 , although the size of each cell might be slightly increased, which is potentially coupled with the accumulation of vesicles in the cytoplasm.
This observation is consistent with our hypothesis that E is loaded into Syt1-positive secretory vesicles in the PG and is released into the hemolymph via exocytosis triggered by calcium signaling. If E indeed requires vesicle-mediated machinery to be released from the PG, there should be transporters on the vesicle surface that load E into the vesicles.
There is an ATP-binding cassette ABC transporter, E23, which has been proposed to function as a 20E exporter to modulate the effective intracellular concentration of 20E in peripheral tissues in Drosophila Hock et al. E23 is a member of the ABCG subfamily of ABC transporters, several of which in mammals have been shown to help efflux cholesterol and other types of steroids such as estrogens and their metabolites Imai et al. Based on these previous findings, an in situ hybridization screening of ABCG transporter genes in Drosophila genome Figure S3 was conducted to identify putative E transporters highly expressed in the PG.
Of these two genes, only knockdown of Atet in the PG showed developmental defects indistinguishable from those of knockdown of calcium-regulated vesicle exocytosis genes Figures 4B—D and were also rescued by E feeding. We next expressed a fluorescent protein-tagged Atet in the PG to visualize its subcellular localization. This resulted in the labeling of both the plasma membrane and Syt1-positive vesicles Figure 4E , suggesting that Atet could indeed be involved in the import of E into these vesicles.
In order to further examine if Atet is a critical transporter for loading E into PG secretory vesicles, we sought to develop an in vitro transport assay. We first analyzed the predicted membrane topology of Atet using Phobius, a transmembrane protein topology and signal peptide predictor program Kall et al.
To our surprise, Atet was predicted to have an extracellular N-terminus, despite the fact that its ATP binding domain is on its N-terminal side Figure 5A. To determine the actual topology of Atet, we expressed an N-terminally HA-tagged Atet in Schneider 2 S2 cells, a cell line derived from a primary culture of late stage Drosophila embryos, and immuno-stained the cells in both permeabilized and non-permeabilized conditions Figure 5B.
Under permeable conditions, both the surface of the cells and the internal structures were stained, suggesting that a certain population of Atet proteins are localized on the plasma membrane as in the PG cells. Importantly, under non-permeable conditions, N-terminal staining of Atet was still detected on the surface of the cells, whereas the control E23 tagged at the intracellular C-terminus was not detected without permeabilizing the cells Figure 5B.
These observations demonstrate that the N-terminus of Atet is indeed located on the non-cytoplasmic side of the membranes. Based on this atypical membrane topology of Atet, we designed an in vitro transport assay using S2 cell membrane vesicle preparations from cells transfected with Atet Figure S4. A crude membrane preparation typically contains both inside-out and right-side-out vesicles. In a regular vesicular ABC transporter assay, only the activity of the transporters in the inside-out vesicle configuration are detected, since a typical ABC transporter in the right-side-out vesicles will have its ABC domain inside the vesicles and therefore unable to access the exogenously added ATP and transport substrate.
Thus, activity is measured as the amount of substrate imported into the vesicles Figure S4. In contrast, in the case of Atet, no net flux into vesicles would be expected upon addition of exogenous substrates since Atet is predicted to pump substrates in the opposite direction. Therefore, in our modified procedure, the substrate E was preloaded into vesicles during isolation and then ATP was added to assess the transporter activity as efflux rather than influx.
These results demonstrate that Atet can indeed transport E from the cytoplasmic to non-cytoplasmic side of vesicle membranes, providing strong support for our vesicle-mediated E release model Figure 6. In the present study, we provide several lines of evidence demonstrating that the insect steroid hormone E is secreted from the PG not by simple diffusion, but rather through a calcium signaling-regulated vesicle fusion event.
Below we discuss three major points of our findings: Atet was originally cloned in Drosophila as an ABC transporter-encoding gene with unknown function. It was found to be highly expressed in embryonic trachea, leading to its name ABC transporter expressed in trachea or Atet Kuwana et al. In our in situ hybridization experiment, however, we found little expression of Atet in embryonic trachea, but instead saw specific high level expression in the PG Figure S3F , consistent with its expression pattern in the third instar larva Figure 4A.
Since we found that Atet has an atypical membrane topology Figures 5A and 5B and can transport E across membranes in vitro Figure 5C , we propose renaming this gene Atypical topology ecdysone transporter , thereby retaining the Atet gene designation. Atet belongs to the ABCG subfamily of ABC transporters, members of which in mammals have been shown to transport cholesterol as well as other steroids, such as estrogens and their metabolites, in many biological systems Imai et al.
To our knowledge, however, the atypical membrane topology, with the N-terminal ABC domain on the non-cytoplasmic side of the membrane, has not been reported for any ABC transporter to date. However, this topology may have a strong advantage in facilitating tight control on E release by preventing Atet from functioning on the plasma membrane, due to the lack of ATP in extracellular space. This configuration therefore prevents E transport directly through the plasma membrane and confines it to a vesicle mediated fusion process, although it requires a separate molecular mechanism to transport ATP into the secretory vesicles.
These transporters mediate cellular cholesterol efflux Wang et al. Clearly, additional studies on the membrane topology of ABCG transporters are warranted. These findings strongly implicate the existence of an unknown GPCR and cognate ligand as mediators of the calcium signaling event that we suggest stimulates E release from the PG.