Use of fluorescently labelled calmodulins as tools to measure subcellular calmodulin activation in living dorsal root ganglion cells.
We have used fluorescently labelled calmodulins to probe the activity of calmodulin in living dorsal root ganglion cells. Calmodulin labelled with the fluorophore 5-([4,6 dichlorotriazin-2yl]amino)-fluorescein (FL-CaM) does not change its fluorescence when it binds calcium, while calmodulin labelled at lysine 75 with 2-chloro-(6-(4-N,N-diethylamino-phenyl)-1,4,5-triazin-4-yl (TA-CaM), an environment-sensitive probe, increases its fluorescence when it binds calcium. We micro-injected FL-CaM or TA-CaM into rat dorsal root ganglion cells and found that both probes localise to the cell nucleus. In contrast, endogenous cellular calmodulin, in dorsal root ganglion cells as in hippocampal neurones, is predominantly cytosolic unless the neurones are depolarised, then it moves to the nucleus. FL-CaM and TA-CaM, introduced into dorsal root ganglion cells via a patch pipette, also immediately move to the nucleus, indicating that the nuclear localisation is a property of the labelled calmodulins. Although the subcellular distribution of FL-CaM and TA-CaM does not necessarily match that of endogenous calmodulin, we show that FL-CaM can be used as a control for TA-CaM when studying calmodulin activation in different cellular compartments.
Milikan
J M
JM
Department of Physiology, University College of London, UK.
Bolsover
S R
SR
D000818:Animals; D002147:Calmodulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D007700:Kinetics; D008845:Microinjections; D018613:Microscopy, Confocal; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D014227:Triazines
C404210:2-chloro-(6-(4-N,N-diethylaminophenyl)-1,4,5-triazin-4-yl)calmodulin; C404209:5-((4,6-dichlorotriazin-2-yl)amino)fluorescein; D002147:Calmodulin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D014227:Triazines; D010727:Phosphoric Diester Hydrolases