Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution.
Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.
Remus
Timothy P
TP
Department of Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
Zima
Aleksey V
AV
Bossuyt
Julie
J
Bare
Dan J
DJ
Martin
Jody L
JL
Blatter
Lothar A
LA
Bers
Donald M
DM
Mignery
Gregory A
GA
D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D019556:COS Cells; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002415:Cats; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D006352:Heart Ventricles / Q000166:cytology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate