Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans.
To study the abundance of specific receptors and other cell surface proteins at synapses, it would be advantageous to specifically label these proteins only when inserted in the plasma membrane. We describe a method that allows to fluorescently label cell surface proteins in live and behaving animals, namely in the nematode Caenorhabditis elegans. Proteins such as subunits of the levamisole sensitive nicotinic acetylcholine receptor (nAChR) were epitope-tagged at their extracellular C-termini, and fluorescent antibodies against those tags were injected into the body fluid. These antibodies specifically labelled synaptic regions on the cell surface of muscles and neurons, and simultaneous use of different tags facilitated co-localization studies. Quantification of the fluorescence is possible, as verified by demonstrating that mutations in ric-3 and unc-38, which cause behavioural resistance to cholinergic agonists, strongly reduce or even abolish nAChR cell surface expression. We also used this method to visualize the extracellular peripheral membrane protein ODR-2, which is related to a neurotoxin-like protein regulating vertebrate neuronal nAChRs. Likewise, fluorescent alpha-bungarotoxin, when injected, bound to certain nAChRs in the pharynx and the nervous system. This showed that, theoretically, any molecular interaction of sufficient affinity may be used to specifically label cell surface structures in live nematodes.
Gottschalk
Alexander
A
Department of Biochemistry, Chemistry and Pharmacy, Institute for Biochemistry, Biocenter N210, Johann Wolfgang Goethe-University, Frankfurt, Germany. a.gottschalk@em.uni-frankfurt.de
Schafer
William R
WR
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004396:Coloring Agents; D000939:Epitopes; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D010243:Paralysis / Q000139:chemically induced; D010609:Pharyngeal Muscles / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism
D002038:Bungarotoxins; D029742:Caenorhabditis elegans Proteins; D004396:Coloring Agents; D000939:Epitopes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D018722:Nicotinic Agonists; C421612:Odr-2 protein, C elegans; D011950:Receptors, Cholinergic; D011963:Receptors, GABA-A; D049452:Green Fluorescent Proteins; D009538:Nicotine