DCGs are released from SCs during mating, activating BMP signalling.

<p><b>A, B</b>. SCs from 6-day-old esgF/O^ts virgin males (A) and from males immediately after mating (B) were fixed and stained with an anti-pMad antibody (red) and DAPI (blue), revealing that the proportion of SCs with detectable nuclear pMad is higher in mated animals. <b>C, D.</b> Immediately after mating, living SCs (D) have less GFP-GPI-labelled DCGs than virgins (C). Image shows a single z-plane of gland stained with Lysotracker Red; not all compartments are in the focal plane. Note that the largest MVBL in (C; arrowhead) contains GFP, probably because of fusion between a DCG compartment and the MVBL [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006366#pgen.1006366.ref017" target="_blank">17</a>]. <b>E</b>. Graph shows proportion of SCs with nuclear pMad in 6-day-old virgin, and mated males (dissected 8 min into mating [Mid] and immediately after mating), and mated males expressing <i>dpp</i>-RNAi and Dad in SCs from eclosion onwards using the <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4</i> driver. <b>F</b>. Graph shows number of GFP-GPI-positive DCG compartments in 6-day-old virgin and mated males (using the <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> driver line; Double is twice mated in 2 h), and at different times after single mating in control SCs. Compartments were also counted in SCs expressing <i>Snap24</i> RNAi post-eclosion in virgins and immediately after mating. Labelled compartments were counted using a complete z-series for each cell. Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP/+; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/+</i> (A, B); <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI/CyO</i> (C, D).***P<0.001, Kruskal-Wallis test, n>15. Scale bar for A-B is 20 μm and C-D is 10 μm.</p

Multiple BMP signalling components regulate nuclear growth in SCs.

<p><b>A.</b> Schematic of the paired male accessory glands (arrows), which pump their contents into the ejaculatory duct (arrowhead) during mating. Left inset shows the epithelial secretory monolayer containing secondary cells (SCs; green) and main cells (MCs), all of which are binucleate. Right inset is a section through the gland revealing the large lumen (asterisk). <b>B.</b> SC (circled) expressing a gene trap for the BMP type I receptor <i>tkv</i>, and stained with an antibody against the BMP type II receptor Wit. These receptors are present both on the plasma membrane (arrowhead) and co-localise in intracellular compartments (arrow). DAPI marks nuclei blue in SCs (asterisks) and surrounding non-expressing MCs. <b>C-F.</b> Accessory glands (AGs) from 6-day-old controls (C) and males expressing RNAis targeting <i>tkv</i> (D) or <i>Mad</i> (F) or expressing the BMP signalling antagonist <i>Dad</i> (E) in adult SCs under the control of esgF/O^ts after temperature shift at eclosion. AGs were dissected, fixed and imaged by confocal microscopy. Glands were stained with DAPI (blue) to mark nuclei and an anti-Fas3 antibody (yellow) to mark cell boundaries. SCs express nuclear GFP, which is also present in the cytosol. Pairs of nuclei from binucleate SCs and MCs are indicated by green and red arrows respectively. <b>G.</b> Bar chart showing SC nuclear size relative to that of MC neighbours for glands expressing different transgenes in SCs under esgF/O^ts control, normalized to the ratio for controls. Note that all manipulations that decrease BMP signalling significantly reduce SC nuclear size. Genotypes for images are: <i>w; PBac[544</i>.<i>SVS-1]tkv</i>^{<i>CPTI002487</i>} (B); <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4</i> combined with no other transgene (C); <i>P[TRiP</i>.<i>JF01485]attP2</i> (III) (D); <i>P[w</i>^<i>+</i> <i>UAS-Dad]</i> (II) (E); <i>P[TRiP</i>.<i>JF01263]attP2</i> (III) (F). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for (B) is 10 μm, for all other images it is 20 μm.</p

Model to explain autocrine regulation of DCG replenishment by BMP signalling in SCs.

<p>Schematics of a single SC immediately before and after mating. (1) In virgin males, Dpp is trafficked to and stored in DCGs. Sporadic release of these DCGs activates BMP signalling, and sustains a basal level of growth, DCG biogenesis and exosome secretion. (2) During mating, about 4 mature DCGs are released (3), resulting in an increase in BMP signalling (4), primarily via an autocrine mechanism and probably in pulses. This stimulates growth, but also increases biosynthesis of new DCG compartments (5; solid arrow), ensuring that the total number of DCGs is fully replenished within 24 h. Dashed arrows highlight other parts of the secretory/endolysosomal system that might be affected by altered BMP signalling. Previous data (Corrigan et al., 2014) and data presented here suggest that long-term elevated BMP signalling enhances endolysosomal trafficking.</p

Mating rapidly accelerates the rate of new DCG compartment formation.

<p><b>A, B</b>. Single z-plane images of SCs from 6-day-old virgin males labelled with GFP-GPI, either co-expressing (B) or not expressing (A) <i>dpp</i>-RNAi. Reducing Dpp signalling decreases the number of labelled DCGs (arrow). <b>C-E</b>. 6-day-old males were shifted to 28.5°C to induce GFP-GPI expression for 16 hrs in virgins (C) or immediately after (D) or before (E) mating. Their AGs were dissected and imaged; each image is from a single z-plane. <b>F</b>. Graph showing number of GFP-GPI-positive DCGs in SCs from either 6-day-old virgin or mated control flies or flies in which BMP signalling is inhibited. There is no decrease in total DCG number in SCs expressing either <i>dpp</i>-RNAi or <i>Dad</i> after mating. <b>G</b>. Graph showing GFP-positive DCG number after a 16 h pulse of GFP-GPI in SCs from 6-day-old males, as in C-E above. Genotypes for images are: <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> combined with no other transgene (A, and pulse-labelled in C, D, E) or <i>P[TRiP</i>.<i>HMS00011]attP2</i> (III) (B). The <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> line was used to generate data in F and G. ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar for A-E, 10 μm.</p

Rapid replenishment of DCGs after mating is BMP-dependent.

<p><b>A</b>. Schematic representation of pulse-chase experiments shown in B-F, indicating the duration of GFP-GPI and <i>Dad</i> overexpression and timings of mating events. <b>B-D</b>. 6-day-old flies were shifted to 28.5°C for 24 h to allow expression of GFP-GPI in virgins (B) or in males mated 8 h after the start of the pulse (C). The number of GFP-GPI-labelled DCGs in SCs was reduced in virgin males co-expressing Dad (D). <b>E.</b> Graph shows a significant increase in the number of labelled DCGs if males are mated at 8h during a 24 h GFP-GPI pulse. The number of DCGs labelled in virgin and mated males is reduced if <i>Dad</i> is co-expressed. <b>F</b>. The increase in labelled compartments after mating is also reduced by Dad co-expression. The <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> line was used to generate data in E and F. *P<0.05, ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar in B-D, 10 μm.</p

Autocrine Dpp regulates SC growth, SV number, endolysosomal trafficking and exosome secretion.

<p><b>A</b>. Expression of <i>dpp</i>-RNAi during the first six days of adulthood using the esgF/O^ts driver reduces the size of SCs and their nuclei (green arrows) relative to MCs (red arrows). <b>B</b>. Relative SC:MC nuclear size for SCs expressing RNAis targeting <i>dpp</i> and <i>gbb</i>, or GFP-tagged Dpp and Gbb, revealing specific effects of Dpp on growth. <b>C</b>. <i>dpp</i>^<i>blk</i>-GAL4 drives expression of a UAS-coupled nuclear GFP exclusively in SCs of the AG. <b>D, E.</b> Mosaic expression of <i>dpp</i>-RNAi or Dpp-GFP in a subset of SCs has a stronger effect on nuclear growth in expressing cells (on–green arrows) than in non-expressing (off–red arrows; white dashed circle) SCs, although <i>dpp</i> knockdown also reduces growth in the latter. <b>F-K.</b> Co-expression of <i>dpp</i>-RNAi with CD63-GFP using the <i>dsx</i>-GAL4 driver (G) reduces non-acidic SV number (eg., marked by arrowhead) and increases GFP fluorescence in largest MVBL (arrow; stained with Lysotracker Red) compared to controls (F); the statistical analysis of these changes for two independent RNAis is shown in H and I respectively. Knockdown of <i>dpp</i> either results in a small increase in the size of the largest MVBL or no significant size change (J), and reduces exosome secretion (K). Confocal images are from fixed glands (A, C, D) stained with DAPI (blue) and for Fas3 (yellow) or from living glands (F, G). Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/P[TRiP</i>.<i>HMS00011]attP2</i> (A and mosaic in D; the esgF/O^ts driver was also used to generate data in E); <i>w; P[w</i>^<i>+</i> <i>UAS-GFP</i>_<i>nls</i><i>]; P[w</i>^<i>+</i> <i>dpp</i>^<i>blk</i><i>-GAL4]</i> (C); <i>w; UAS-CD63-GFP tub-GAL80</i>^<i>ts</i><i>; dsx-GAL4</i> combined with no other transgene (F) or <i>P[TRiP</i>.<i>HMS00011]attP2</i> (III) (G). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for A, D is 20 μm, F, G, 10 μm, and for C, 50 μm.</p

Dpp is stored in SC dense-core granules.

<p><b>A</b>. Live image of Dpp-GFP-expressing SC stained with Lysotracker Red to identify acidic compartments. Note that Dpp-GFP localises to spherical structures (arrows) that are distinct from acidic compartments. <b>B</b>. Image of fixed SC, stained with anti-ANCE antibody (red) and DAPI (blue) after 24 h pulse of Dpp-GFP expression, reveals co-localisation of GFP fluorescence with ANCE-positive DCGs (arrows). <b>C</b>. Rab11-YFP-positive compartments in SCs from flies ubiquitously expressing this fusion protein under tubulin promoter control contain DCGs that stain positive for ANCE (red). <b>D</b>. Fixed SC expressing GFP-GPI and stained with anti-ANCE (red) and DAPI (blue), showing co-localisation of ANCE and GFP in DCGs (arrows). <b>E</b>. GFP-positive puncta (arrow) and filaments (arrowhead) are detected in the AG lumen when Dpp-GFP is expressed in SCs. <b>F, G.</b> SC-specific expression of <i>Snap24</i> RNAi in adults significantly increases number of GFP-GPI-labelled DCGs in 6-day-old virgin males (F; using <i>spi</i>-GAL4 driver), and reduces nuclear size (G; using esgF/O^ts driver). All images are from 3-day-old virgin males and individual SCs are outlined by dashed circles. Genotypes for images are: <i>w; tub-GAL80</i>^<i>ts</i><i>; dsx-GAL4/UAS-Dpp-GFP</i> (A, B, E); <i>w; tub-rab11-YFP</i> (C); <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI/CyO</i> (D). ***P<0.001, Mann-Whitney <i>U</i> test, n = 10. Scale bar for A-D is 10 μm and E is 20 μm.</p