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

Summary

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

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.

Introduction

All internal organs are composed of tubes, crucial for their many different functions, such as the transport and exchange of gases, liquids and nutrients and the excretion of metabolic waste. Their polarized character, with distinct apical and lumenal membranes, is adapted to these specific functions, and defects in the biogenesis of their endo- and plasma membrane systems are a frequent cause of human disease^1,2. The majority of tubes of the vasculature and of internal organs are multicellular and form a lumen intercellularly; however, unicellular tubes, which form the lumen intracellularly, can, for example,....

Protocol

1. Labeling the C. elegans Excretory Canal by Fluorescent Fusion Proteins¹⁴

Note: See the accompanying paper on intestinal tubulogenesis³ for labeling by in situ antibody staining procedures adaptable to the excretory canal. See Table 1 for examples of molecules proven useful for visualizing C. elegans excretory canal endo- and plasma membranes, Table 2 for examples of promoters driving expression to the excretory canal, and Table 3 for resources for more comprehensive collections of markers and promoters, including re....

Results

This protocol describes how to use the C. elegans excretory canals to visually and molecularly analyze unicellular tubulogenesis and intracellular lumen morphogenesis in a single cell. During their extension from the time of mid-embryogenesis to adulthood, the four excretory canals continue to expand their basolateral and apical/lumenal membranes together with their canalicular and endosomal endomembrane system, providing a unique model for the in vivo analysis of de.......

Discussion

C. elegans’ genetic versatility, transparency, simple body plan and invariant cell lineage all make it an excellent model for the analysis of morphogenesis. This protocol describes how to combine standard genetic manipulations and imaging studies to take advantage of the 2 micron thin C. elegans excretory canals to study polarized membrane and intracellular lumen biogenesis in a single cell tube.

Labeling
The C. elegans excretory canals c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cloning
Plasmid pPD95.75	Addgene	Cat. No. 37464
PCR Kit	Qiagen	Cat. No. 27106
Ligation kit	New England Biolabs	Cat. No. E2611L
DNA marker	Thermo Scientific	Cat. No. SM1331
Agarose DNA grade	Fisher Scientific	Cat. No. BP164-100
Competent cells	New England Biolabs	Cat. No. C2987H
Tris	Fisher Scientific	Cat. No. BP154-1
EDTA	Sigma	Cat. No. ED-1KG
Acetic acid	Fisher Scientific	Cat. No. A38S-500
Ethidium bromide	Fisher Scientific	Cat. No. BP1302-10
Equipments
PCR machine	MJ Research	Cat. No. PTG-200
Centrifuge	Eppendorf	Cat. No. 5415C
Water Bath	Precision Scientific	Cat. No. 666A3
Gel running instrument	Fisher Scientific	Cat. No. 09-528-165
Gel running power supply	Fisher Scientific	Cat. No. 45-000-465
Molecular Imager Gel Doc XR System	Bio-Rad	Cat. No. 1708195EDU
Nanodrop Spectrophotometer	Thermo Scientific	Cat. No. ND1000
C. elegans related1			1see reference19 for standard C. elegans culture and maintenance procedures.
LB Medium and plates2			2see reference19 for protocols.
Tryptone	Acros Organics	Cat. no. 611845000
Yeast Extract	BD Biosciences	Cat. no. 212750
NaCl	Sigma	Cat. no. S7653
Bacto Agar	BD Biosciences	Cat. no. 214040
Ampicillin	Sigma	Cat. no. A0116
Tetracycline	Fisher Scientific	Cat. no. BP912
M9 Medium2			2see reference19 for protocols.
NaCl	Sigma	Cat. no. S7653
KH2PO4	Sigma	Cat. no. P0662
Na2HPO4	Sigma	Cat. no. S7907
MgSO4	Sigma	Cat. no. M2773
NGM plates 2			2see reference19 for protocols.
NaCl	Sigma	Cat. no. S7653
Peptone	BD Biosciences	Cat. no. 211677
Tryptone	Acros Organics	Cat. no. 611845000
Bacto Agar	BD Biosciences	Cat. no. 214040
MgSO4	Sigma	Cat. no. M2773
CaCl2	Sigma	Cat. no. C3881
Cholesterol	Sigma	Cat. no. C8667
K2HPO4	Sigma	Cat. no. P3786
KH2PO4	Sigma	Cat. no. P0662
RNAi plates3			3see reference21 for protocols.
NaCl	Sigma	Cat. no. S7653
Peptone	BD Biosciences	Cat. no. 211677
Tryptone	Acros Organics	Cat. no. 611845000
Bacto Agar	BD Biosciences	Cat. no. 214040
MgSO4	Sigma	Cat. no. M2773
CaCl2	Sigma	Cat. no. C3881
Cholesterol	Sigma	Cat. no. C8667
K2HPO4	Sigma	Cat. no. P3786
KH2PO4	Sigma	Cat. no. P0662
IPTG	US Biological	Cat. no. I8500
Carbenicillin	Fisher Scientific	Cat. no. BP2648
NaOH	Fisher Scientific	Cat. no. SS266-1
Sodium hypochlorite	Fisher Scientific	Cat. no. 50371500
Bacteria
OP50 bacteria	CGC
HT115 bacteria	CGC
Genome-wide RNAi libraries
Ahringer genome-wide RNAi feeding library (ref21,58,59)	Source BioScience
C. elegans ORF-RNAi feeding library (ref60)	Source BioScience
Imaging related
Lidocaine	MP Biomedicals,LLG	Cat. no. 193917
Materials
Vacuum Grease Silicone	Beckman	Cat. no. 335148
Microscope slides	Fisher Scientific	Cat. no. 4448
Tissue culture plate, 6 well	Corning Inc.	Cat. no. 08-772-33
Equipment
SMZ-U dissecting microscope (Nikon)
SZX12 dissecting microscope (Olympus), equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions).
TCS SL Laser-scanning confocal microscope (Leica Microsystem)
C2 laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope (Nikon)