title: 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.
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Linda S
authors.initials: LS
authors.lastname: Nikolova
authors.affiliation: Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA markm@genetics.utah.edu.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Metzstein
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology*; D004330:Drosophila / Q000254:growth & development*; D029721:Drosophila Proteins / Q000378:metabolism*; D005614:Freeze Fracturing; D042541:Intracellular Space / Q000502:physiology*; D007814:Larva / Q000254:growth & development / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D029721:Drosophila Proteins; C543875:Rbcn-3B protein, Drosophila; D025262:Vacuolar Proton-Translocating ATPases
keywords: CLEM; Drosophila; Freeze substitution; HPF; Lumenogenesis; TEM