Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

Abstract

Apoptosis, also called programmed cell death, is cellular suicide in multicellular organisms [1][2][3]. The caspases are a family of cysteine proteases that play a critical role in the execution phase of apoptosis.They are responsible for the biochemical and morphological changes associated with apoptosis [4]. It has been proposed that caspases can be divided into two categories: "initiator" caspases with a long prodomain, such as caspase-9, and "effector" caspases such as caspase-3[5]. Caspase-3 has been shown to be a key component involved in the underlying mechanisms of apoptosis [6], and the activation of caspase-3 is considered to be the final step in many apoptotic pathways [7]. Moreover, the combination of FCCS and three colors tandem fused fluorescent proteins can predict progression of the cell to apoptosis before the morphologic changes appeared. transfected with 0.5 μg/ml for 6-well, or 0.2 μg/ml for 8-well of plasmid DNA encoding the CRY chimera using Optifect TM (Invitrogen). Materials and methods Plasmids Induction and inhibition of apoptosis. For induction of apoptosis,the CRY chimera-expressing HeLa cells were incubated with 250 ng/ml TNF-α and 20 μg/ml CHX in humidified air with 5% CO 2 at 37°C. To specifically inhibit caspase-3 or caspase-9, the CRY chimeraexpressing HeLa cells were treated with 5 μM caspase-3 inhibitor II or 25 μM caspase-9 inhibitor I two hours before addition of TNF-α/CHX.Preparation of cell lysates. The CRY chimera-expressing HeLa cells were lysed with lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1%Triton X-100) [26] at 0 min, 30 min and 60 min after TNF-α/CHX treatment. 7FCCS measurement and data analysis. FCCS measurements were performed with an LSM510-ConfoCor3 (Carl Zeiss), the setup of which followed our previous report [23]. ECFP and Venus were excited at the 458 nm laser line because the brightness of Venus was sufficient in this case for FCCS measurement, and mCherry was excited at the 594 nm laser line.These lines were via a single-band dichroic mirror for 458 nm, which where and particles that have both red and cyan fluorescence (N cross(CR) ), or red and yellow fluorescence (N cross(RY) ) can be calculated. Here, the RCA Mix value reported previously was used for cell lysates [23]. Results TNF-α/CHX-induced time-dependent activation of caspases in HeLa cell lysatesIn this study a fusion protein was constructed and named CRY chimera.As shown in Fig. 1A, the CRY chimera was formed by inserting DEVD and LEHD amino acids between ECFP and mCherry and mCherry and Venus, respectively. The CRY chimera-expressing HeLa cells are shown in Fig. 1B. DEVD sequences between ECFP and mCherry and LEHD sequences between 10 mCherry and Venus could be cleaved by the activated caspase-3 and caspase-9, respectively. The cross-correlation of CRY chimera was observed in fluorescent protein pairs ECFP and mCherry (C-DEVD-R) and mCherry and Venus (R-LEHD-Y) in HeLa cells (Fig. 1C and D). To evaluate the activation of caspase-3 and caspase-9, FCCS measurements were carried out i...

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

“…For the semi-quantitative evaluation of cross-correlations the relative cross-amplitude (RCA) was calculated using equation (11). Figure S1A (14) and (15).…”

“…FCCS provides information about the coincidence of two spectrally well-defined fluorescent molecules in a small detection volume with singlemolecule sensitivity [11]. The femtoliter confocal volume is well suited to resolve the different measurement positions even in live cells.…”

miRNA and nasopharyngeal carcinoma

“…Plk1 depletion induces significant G2/M arrest and apoptosis mediated by caspase 3/7 activation [58]. Radiation enhances G2/M arrest and apoptosis [59], so the combination of radiation and siPlk1 may delay tumor formation [60].…”

Heterosídeos alcaloídicos de Solanum lycocarpum A. St.-Hil.: avaliação das atividades contra fungos dermatófitos e câncer de pele

“…As caspases, por sua vez, são uma família de proteases cisteínicas que desempenham um papel importante na apoptose, pois são as responsáveis por alterarem a morfologia e bioquímica das células (COHEN, 1997;MIKUNI;KINJO, 2011). Existem dois tipos de caspases segundo Sun et al (1999): as que são "iniciadoras" da apoptose, como é o caso das caspase -8 e -9, e as "efetoras", no caso das caspases-3, -6 e -7.…”

“…A caspase-3 é uma protease cisteínica efetora de grande importância na apoptose, pois é a responsável pela clivagem proteolítica total e parcial de proteínas, como a enzima nuclear poli(ADP-ribose) polimerase (PARP), a qual é clivada por diferentes vias durante a apoptose celular (COHEN, 1997). Dessa maneira, caspase-3 tem demonstrado ser um componente chave envolvido nos mecanismos de apoptose, sendo que sua ativação é considerada como ponto final de muitas vias apoptóticas (SUN; MIKUNI; KINJO, 2011). De acordo com os resultados obtidos na contagem de células marcadas por caspase-3, verificouse que o grupo que obteve redução tumoral, que foi tratado com a formulação Curaderm BEC5, apresentou maior número de células em apoptose (Figura 23).…”