Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment

Abstract

Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae.

Time to make the doughnuts: Building and shaping seamless tubes

“…These vesicles are proposed to coalesce to form the intracellular apical domain, which then connects to other tubes via anastomosis [11, 22, 44, 55]. This mechanism of tube formation is sometimes called “cell hollowing” (Fig.…”

“…Various large intracellular vacuoles or multivesicular bodies (MVBs) have been detected near the growing lumen of vertebrate capillaries [9, 11], Drosophila terminal cells [55] and the C. elegans canal cell [4, 58], and have been speculated to derive from pinocytosis and contribute to the apical membrane. However, the origin of these vacuoles has not been determined definitively.…”

“…The V-ATPase is a proton pump involved in vesicle acidification; it plays multiple roles in vesicle trafficking, fusion and lysosomal degradation [83]. Both C. elegans and Drosophila V-ATPase mutants accumulate excess MVBs, consistent with a trafficking defect, but the V-ATPase restrains lumen growth in the canal cell [55, 81, 82, 84], yet promotes lumen growth in terminal cells [52, 55]. The mechanistic basis for these discrepancies is not known, but they could reflect differences in MT orientation (Figs.…”

“…The mechanistic basis for these discrepancies is not known, but they could reflect differences in MT orientation (Figs. 2C, 3D), differences in junction organization at the cell base [25], or differences in the types of vesicles that contribute to the lumen of each cell [55]. …”

Faithful mRNA splicing depends on the Prp19 complex subunit faint sausage and is required for tracheal branching morphogenesis in Drosophila

“…Unless noted otherwise, Drosophila stocks are described in FlyBase and were obtained from the Bloomington Stock center: btl 724 (Ghabrial and Krasnow, 2006) (Guilgur et al, 2014); UAS-bnl ; UAS-λBtl (Lee et al, 1996); UAS-palm-mNeonGreen (this study); UAS-CG17716 (this study); btl-Gal4 (Shiga et al, 1996); Abd-B-Gal4 (de Navas et al, 2006); btl-Gal80 (Nikolova and Metzstein, 2015); da-Gal4; and 69B-Gal4. fas …”