On-line monitoring of apoptosis in insulin-secreting cells.
Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.
Köhler
Martin
M
Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden. martin.kohler@molmed.ki.se
Zaitsev
Sergei V
SV
Zaitseva
Irina I
II
Leibiger
Barbara
B
Leibiger
Ingo B
IB
Turunen
Mikael
M
Kapelioukh
Iouri L
IL
Bakkman
Linda
L
Appelskog
Ioulia B
IB
de Monvel
Jacques Boutet
JB
Imreh
Gabriela
G
Berggren
Per-Olof
PO
D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D020407:Internet; D007515:Islets of Langerhans / Q000378:metabolism*; D008164:Luminescent Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008991:Monitoring, Physiologic; D009765:Obesity / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D017785:Photons
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D007202:Indicators and Reagents; D007328:Insulin; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases