The course of major developmental events in the late larval (in late 3^rd instar larva) and prepupal salivary glands illustrated by staining with antibodies to highlight appropriate structures.

<p>(<b>a</b>) At -12 hr prior to pupariation, when Sgs glue proteins and secretory granules are synthesized, a dense “reticulate”meshwork forms from cytoskeletal components inside cells; myosin II (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>b</b>) During metamorphic pulse of ecdysteroids at 7 hrs prior to pupariation (-7 hr), the larval salivary glands start to release the accumulated secretory granules into the lumen by exocytosis; transcription factor BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>c</b>) At -3 hr prior to pupariation (-3 hr), exocytosis is complete and the salivary gland undergoes glue solvatation, increasing the diameter of the lumen. This solvatation will facilitate the expectoration of the glue at the pupariation; myosin II (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>d</b>) About +2 hr APF, the salivary gland cells become highly vacuolized by membrane recycling due to massive endocytosis, a consequence of exocytosis; BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>e</b>) The process of vacuolization and membrane recycling is consolidated by +7 hr APF, shortly prior to the next secretion; BR-C (red), p127^l(2)gl (green) and filamentous actin (blue). (<b>f</b>) At +8 hr APF, the salivary glands are showing an early phase of release of myosin II, p127^l(2)gl and filamentous actin into the centrally located lumen. fb in (<b>a</b>), (<b>b</b>), (<b>d</b>)  =  piece of adherent fat body. All confocal images 400×.</p

A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by <i>lacZ</i>-containing <i>P</i>-element insertion stocks.

<p>We consistently used 9–10 hr old prepupal salivary glands for these types of detection. (<b>a</b>) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF₄₈₈-phalloidin for F-actin. (<b>b</b>) Similarly to (<b>a</b>), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). (<b>c</b>) Tumor suppressor protein p127, the product of <i>l(2)gl</i> gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 (<b>d</b>). Examples of proteins monitored via <i>lacZ</i>-fusion include the transcription factor Ttk (<b>e</b>), the dual-specific LAMMER kinase Doa (<b>f</b>), the D subunit of the vacuolar H⁺ vATPase Vha36-1 (<b>g</b>) and the transcription factor Fkh (<b>h</b>).</p

Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen.

<p>Panels a and b show western blots of secreted proteins isolated from the lumen. (<b>a</b>) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). (<b>b</b>) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). (<b>c</b>) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. (<b>d</b>) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. (<b>e</b>) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. (<b>f</b>, <b>g</b>) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells (<b>f</b>), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen (<b>g</b>); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t001" target="_blank">Tables 1</a> through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t004" target="_blank">4</a>. In addition, <i>in situ</i> hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae (<b>h</b>, <b>i</b>, (green)) along with F-actin (<b>h</b>, <b>j</b>, (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When <i>in situ</i> hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene <i>Doa</i> locus, signal was found only in nuclei (<b>k</b>, <b>n</b>, (red)) together with Hoechst 33258 staining DNA (<b>k</b>, <b>l</b>, (green)), while F-actin was detectable in the lumen (<b>k</b>, <b>m</b>, (blue)). Remaining confocal images 400×. L in (<b>f</b>), (<b>g</b>), (<b>h</b>) and (<b>k</b>)  =  lumen.</p

Scanning electron microscopic images of the apocrine process in the 9

<p>The gland, dissected under the stereomicroscope and having a lumen evidently filled with material, was fixed and processed to critical point drying, after which it was broken up to expose inferior portion that included the luminal surface, and then sputter coated. The image reveals (<b>a</b>) numerous aposome-like spheres (arrows) and various material-bearing structures on the surface of apical membrane (10000×). In addition, at higher magnification (<b>b</b>), some of these spheroid structures (arrows) displayed constrictions and show a decapitation of the aposome's stalk (arrowheads) (20000×).</p

Evidence for the graded temporal release of different proteins by apocrine secretion.

<p>(<b>a</b>) = At+8.5 hr APF, the ribosomal protein Rp40 (blue) is completely released into lumen, the cortical membrane component α-spectrin (green) was removed from the lateral and apical surfaces but remained at the basal surface, and the nuclear receptor Usp (red) is about half-released into the lumen. (<b>b</b>) At +9 hr APF, both the ribosomal protein Rp21 (green) as well as the ecdysone-inducible Ets-like E74 transcription factor (red) are present only in the lumen, whereas there remains significant F-actin (blue) signal on the cortical membranes. (<b>c</b>) At the same time (+9 hr APF), the ecdysone-regulated transcription factor and nuclear tumor suppressor are secreted differently: while Kr-h (red (<b>d</b>)) is completely extruded into the lumen, p53 (green (<b>e</b>)) only starts to be released and the majority of its signal is still detected in nuclei. Although filamentous actin (blue (<b>f</b>)) already is being secreted into the lumen, there is detectable signal still visible on cell membranes. (<b>g</b>) During +9 to +10 hr APF, the ecdysone-regulated transcription factor BR-C (green (<b>h</b>)) is completely released into the lumen, whereas lamin C (red), a component of the nuclear envelope, is only partially released and can be still detected on the nuclear membrane (<b>i</b>). Although filamentous actin (blue) is already within the lumen, significant amounts of it still line the cortical cytoskeleton, mainly at the apical membrane (<b>j</b>). (<b>k</b>) At the end of +10 hr APF both, Rab11 (green (<b>l</b>)), a member of the GTPase family of membrane proteins as well as the tumor suppressor transcription factor p53 (red (<b>m</b>)) have been completely secreted into the lumen. Hoechst 33258 was used to detect nuclear DNA (blue (<b>n</b>)) which stays in nuclei. All confocal images 400×.</p

List of proteins released by apocrine secretion and detected by antibodies using immunostaining.

<p>This table shows 47 proteins identified using laser confocal or fluorescence microscopy of antibody-stained salivary glands. Proteins are listed alphabetically with the corresponding gene name, molecular weight (in kDa), function and predominant cellular localization. The rightmost columns describe the detection method and predominant time of their release into lumen.</p

List of proteins released by apocrine secretion and detected by fluorescent tagging.

<p>Table shows 32 proteins identified using GFP-/EYFP-/RFP-constructs, as mentioned also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#s2" target="_blank">Materials and Methods</a> section. Also here proteins are listed alphabetically with the corresponding gene name, molecular weight (in kDa), function and predominant cellular localization. The rightmost columns describe not only the detection method but also predominant time of their release into lumen and whenever possible also genotype reference.</p><p>*non-FBti and non-FBal References related to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t003" target="_blank">3</a>.</p><p>{a} Flytrap (<a href="http://flytrap.med.yale.edu/" target="_blank">http://flytrap.med.yale.edu/</a>).</p><p>Morin X, Daneman R, Zavortink M and Chia W (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in <i>Drosophila</i>. Proc. Natl. Acad. Sci. USA 98: 15050–15055.</p><p>{b} Gavdos Protein trap.</p><p>(<a href="http://biodev.obs-vlfr.fr/gavdos/protrap.htm" target="_blank">http://biodev.obs-vlfr.fr/gavdos/protrap.htm</a>) Alain Debec; Biologie du Développement, UMR 7009, CNRS/Université Pierre et Marie Curie, Observatoire Océanologique, Villefranche sur mer, 06230, France.</p><p>{c} Kanesaki T, Edwards CM, Schwarz US and Grosshans J (2011) Dynamic ordering of nuclei in syncytial embryos: a quantitative analysis of the role of cytoskeletal networks. Integr. Biol. (Camb.) 3: 1112–1119.</p><p>{d} Edwards KA, Demsky M, Montague RA, Weymouth N and Kiehart DP (1997) GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in <i>Drosophila</i>. Dev. Biol. 191: 103–117.</p><p>{e} Costantino BF, Bricker DK, Alexandre K, Shen K, Merriam JR, Antoniewski C, Callender JL, Henrich VC, Presente A and Andres AJ (2008) A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of <i>Drosophila</i>. PLoS Genet. 4: e1000102.</p

Transmission electron microscopy reveals an apocrine process in 8–10 hr old prepupal salivary glands.

<p>(<b>a</b>) <i>Prima vista</i> evidence of apocrine secretion is documented by apical protrusions (arrows) and numerous cytoplasmic fragments (arrowheads) inside lumen of the salivary glands from +9 hr APF animal; 2700×. Higher magnification views (<b>b</b> and <b>c</b>) of the apocrine process showing details of electron-dense material (arrows) released from the apical surface (arrowheads) of 9-hr old prepupal salivary gland cells; 8000× and 10000×, respectively. However, at the very early phases of apocrine secretion, +8 hour APF, the salivary gland cells show prominent and numerous microvilli (m) and the lumen is filled with “uncertain” whorled membraneous-like (arrows) (<b>d</b>) or electron-translucent filament-like material (<b>e</b>); both 2700×. Slightly later (+8.5 hr APF), the apical surface of the cells still contains numerous microvilli (m), but the material inside the lumen becomes electron dense and almost evenly distributed (arrows), consisting of many small pieces (<b>f</b>); 4000×. At the mid-phase of apocrine secretion (+9 hr APF), microvilli (m) are less abundant (arrows), and larger pieces and more electron dense material (arrowheads) start to appear in the lumen (<b>g</b>); 6700×. At later stages of apocrine secretion (+10 hr APF), the microvilli are absent and the luminal material becomes flocculent; it stays electron-dense, and larger pieces of material (arrows) are irregularly scattered in the lumen. Some of these clearly contain structured material of the cytoplasm including ER, Golgi (G), mitochondria (M) or multivesiculated elements (MVE) (<b>h</b>, <b>i</b>, <b>j</b>); 2700×, 8000× and 14000×, respectively. L in all images means lumen.</p

List of proteins released by apocrine secretion and detected by chromogenic staining.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t003" target="_blank">Table 3</a> shows 44 entities detected by positive LacZ staining of <i>P</i>-element insertions, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#s2" target="_blank">Materials and Methods</a>. Also these proteins are listed alphabetically with the corresponding gene name, molecular weight (in kDa), function and predominant cellular localization. The rightmost columns describe not only the detection method but also predominant time of their release into lumen and whenever possible also genotype reference.</p><p>*non-FBti and non-FBal References related to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094383#pone-0094383-t003" target="_blank">3</a>.</p><p>{f} Crispi S, Giordano E, D‘Avino PP, Peluso I and Furia M (2001) Functional analysis of regulatory elements controlling the expression of the ecdysone-regulated <i>Drosophila ng-1</i> gene. Mech. Dev. 100: 25–35.</p

Immunological evidence for massive release of proteins in the salivary glands of 8–10 hr old prepupae.

<p>(<b>a</b>) +8 hr APF: There is an early phase of release of myosin II (red) and β-tubulin (green), while filamentous actin(blue) has become highly accumulated at the apical membrane; (<b>b</b>) +8.5 hr APF: Although α-catenin (blue) and nuclear Smrter (green) have already been completely released into lumen, transcription factor BR-C is still present in some nuclei (red). (<b>c</b>) +9 hr APF: At the mid-phase of secretion, BR-C (red) is mostly released into the wider lumen while cytosolic Rop (green) and filamentous actin (blue) are still mostly retained at their normal cellular locations. (<b>d</b>) +9.5 hr APF: The entire immunohistochemically detectable pool of filamentous actin (blue), myosin II (green) and nuclear Rpd3 (red) become visible only in the lumen. (<b>e</b>) +10 hr APF: The previously released filamentous actin (blue) become undetectable, and ribosomal protein Rp21 (green) and nuclear receptor EcR (red) are solely detected in the lumen. (<b>f</b>) +10.5 hr APF: The lumen has been emptied, and filamentous actin (blue) starts to be detected again only on basal surface. Although the salivary gland was stained also for the presence of Rop (green) and transcription factor BR-C (red), these proteins were not detected. (<b>g</b>) +11 hr APF: by this time, in addition to filamentous actin (blue) being visible on the basolateral membranes and slightly detectable at the apical surface, BR-C (red) begins to be detected again in nuclei. We speculate that th e low red cytoplasmic signal could represent freshly synthesized BR-C prior to its being imported in nuclei. However, Rop (green) is not yet detected by this time. fb in (<b>a</b>), (<b>b</b>), (<b>c</b>), (<b>d</b>)  =  piece of adherent fat body. Described massive protein secretion is accompanied by the relocation of measurable fluorescence from salivary gland cells to the extracellular gland lumen during 8 to 10 hr APF (<b>h</b>). The intracellular <i>vs</i>. lumenal distribution of representative proteins (p127: blue (intracellular) <i>vs</i>. green (lumenal), β-tubulin: yellow (intracellular) <i>vs</i>. magenta (lumenal)) was quantified by measuring the fluorescence signal [Cy5 (633 nm) for β-tubulin; Cy3 (546 nm) for p127; fluorescence intensity was evaluated by using Histogram module of Zeiss AIM LSM5 application] associated with a protein at hourly intervals following pupariation from each of 5 independent glands. All confocal images 400×.</p