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 the 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 methods should be applicable to analyzing the many cell biological problems which can be addressed using Drosophila larvae.
- Received July 6, 2015.
- Accepted September 17, 2015.