Technical Reference #1706

1706.

Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins Jerry E. Chipuka; John C. Fisherb; Christopher P. Dillona; Richard W. Kriwackib;c; Tomomi Kuwanad;1; and Douglas R. Greena;1, St. Jude Children's Research Hospital, PNAS, 105(1706), (2008)
Link To Paper

Abstract:
Normal cellular lifespan is contingent upon preserving outer mitochondrial membrane (OMM) integrity as permeabilization promotes apoptosis. BCL-2 family proteins control mitochondrial outer membrane permeabilization (MOMP) by regulating the activation of the pro-apoptotic BCL-2 effector molecules BAX and BAK. Sustainable cellular stress induces proteins (e.g. BID BIM and cytosolic p53) capable of directly activating BAX and/or BAK but these direct activators are sequestered by the anti-apoptotic BCL-2 proteins (e.g. BCL-2 BCL-xL and MCL-1). In the event of accumulated or marked cellular stress a coordinated effort between previously sequestered and nascent BH3-only proteins inhibits the anti-apoptotic BCL-2 repertoire to promote direct activator proteinmediated MOMP. We examined the effect of ABT-737 a BCL-2 antagonist and PUMA a BH3-only protein that inhibits the entire anti-apoptotic BCL-2 repertoire with cells and mitochondria that sequestered direct activator proteins. ABT-737 and PUMA cooperated with sequestered direct activator proteins to promote MOMP and apoptosis which in the absence of ABT-737 or PUMA did not influence OMM integrity or cellular survival. Our data show that the induction of apoptosis by inhibition of the anti-apoptotic BCL-2 repertoire requires ‘‘covert’’ levels of direct activators of BAX and BAK at the OMM.

Keywords:
BCL-2 family; mitochondrial; MOMP; PUMA

Materials & Methods:
Microinjection and Confocal Microscopy. Cells were plated on glass bottom dishes (MatTek) the day before injection in DMEM (containing: 200 mM L-glutamine 10% heat inactivated FBS and 20 M Q-VD-OPh) and incubated at 37 °C with 5% CO2. At the time of injection cells were approximately 90% confluent. Cells were coinjected with Texas Red dextran (0.2 g/ l 10 kDa Invitrogen Molecular Probes) in HE buffer (10mMHepes pH 7.4 1 mM EDTA); the needle concentrations were: 0.12 g/ l BID (full-length and caspase 8 cleaved) BCL-xL C (0.55 g/ l) and PUMA (0.475 g/ l). Approximately 10–50 femtoliters are injected per cell; the range is due to the back pressure within the cell which is dictated by where in the cytoplasm the needle enters. The average cell is approximately 1 nl in volume so an injection of 10–50 fl equals an approximate range of 0.054–0.27 nM C8-BID 0.37–1.85 nM BCL-xL C and 0.25–1.25 nM PUMA . Cells were injected using an Eppendorf InjectMan NI2/FemtoJet microinjection system (Brinkmann Instruments) mounted on a Nikon TE2000-U inverted microscope (Nikon USA) with an ELDW 40X Plan Fluor phase-contrast objective (NA 0.6). Cells were kept in the stage for less than 20 min at a time. Injection parameters: Pi: 30–120 hPa Ti: 0.5 s Pc: 20 hPa. Immediately after injection cells were allowed to recover at 37 °C 5% CO2. Confocal microscopy on live cells was performed with a Marianas spinning disk confocal imaging system (Intelligent Imaging Innovations/3i) consisting of a CSU22 confocal head (Yokogowa Electric Corporation) DPSS lasers (Crysta- Laser) with wavelengths of 445 nm 473 nm 523 nm 561 nm and 658 nm and a Carl Zeiss 200M motorized inverted microscope (Carl Zeiss MicroImaging) equipped with spherical aberration correction optics (3i). Temperature was maintained at 37 °C and 5% CO2 using an environmental control chamber (Solent Scientific). Images were acquired with a Zeiss Plan-Neofluar 40 1.3 NA DIC objective on a CascadeII 512 EMCCD (Photometrics) using SlideBook 4.2 software (3i).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HeLa