title: Genetically encoded far-red fluorescent sensors for caspase-3 activity.
abstract: Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues.
authors: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors: Olga A
authors: OA
authors: Zlobovskaya
authors: Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
authors: Tatiana F
authors: TF
authors: Sergeeva
authors: Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
authors: Marina V
authors: MV
authors: Shirmanova
authors: Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.|Nizhny Novgorod State University, Nizhny Novgorod, Russia.
authors: Varvara V
authors: VV
authors: Dudenkova
authors: Faculty of Medicine, Lomonosov Moscow State University, Moscow, Russia.
authors: George V
authors: GV
authors: Sharonov
authors: Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia.
authors: Elena V
authors: EV
authors: Zagaynova
authors: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
authors: Konstantin A
authors: KA
authors: Lukyanov
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015374:Biosensing Techniques; D053148:Caspase 3 / Q000032:analysis* / Q000378:metabolism; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D000097573:Red Fluorescent Protein
chemical_list: D053148:Caspase 3; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: FRET; apoptosis; bacteriophytochrome-based infrared fluorescent protein; caspase-3; genetically encoded fluorescent sensor; green fluorescent protein (GFP)