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.