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The C. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: Labeling by GFP-fusions, RNAi interaction screen and imaging
Zhang N.2; Membreno E.2; Raj S.2; Zhang H.2; Khan L.A.2; Gobel V.2
2017-10-03
Source PublicationJournal of Visualized Experiments
ISSN1940087X
Volume2017Issue:128
Abstract

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

KeywordConfocal Microscopy Developmental Biology Developmental Genetics Dissecting Fluorescence Microscopy Intracellular Lumen Morphogenesis Issue 128 SIngle Cell In Vivo Analysis Unicellular Tubulogenesis
DOI10.3791/56101
URLView the original
Indexed BySCI
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000415371500029
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Cited Times [WOS]:1   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Affiliation1.Universidade de Macau
2.Massachusetts General Hospital
3.Jilin University
Recommended Citation
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Zhang N.,Membreno E.,Raj S.,et al. The C. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: Labeling by GFP-fusions, RNAi interaction screen and imaging[J]. Journal of Visualized Experiments,2017,2017(128).
APA Zhang N.,Membreno E.,Raj S.,Zhang H.,Khan L.A.,&Gobel V..(2017).The C. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: Labeling by GFP-fusions, RNAi interaction screen and imaging.Journal of Visualized Experiments,2017(128).
MLA Zhang N.,et al."The C. elegans excretory canal as a model for intracellular lumen morphogenesis and in vivo polarized membrane biogenesis in a single cell: Labeling by GFP-fusions, RNAi interaction screen and imaging".Journal of Visualized Experiments 2017.128(2017).
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