Technical Reference #1667

1667.

Caspase Cleavage of Atg4D Stimulates GABARAP-L1 Processing and Triggers Mitochondrial Targeting and Apoptosis Virginie M. S. Betin and Jon D. Lane, University of Bristol, Journal of Cell Science, 122(1667), (2009)
Link To Paper

Abstract:
Autophagy is an important catabolic process with roles in cell survival and cell death. It sequesters cytosol and organelles within double-membrane autophagosomes that deliver their contents to lysosomes for degradation. Autophagosome biogenesis is coordinated by the autophagy-related protein 4 (Atg4) family of C54 endopeptidases (Atg4A-Atg4D). These enzymes prime and then later delipidate the autophagosome marker Atg8. Here we show that one family member Atg4D is cleaved by caspase-3 in vitro and in apoptotic cells. Atg4D is a poor priming and delipidation enzyme in vitro but truncated N63 Atg4D displays increased activity against the Atg8 paralogue -aminobutyric acid receptor-associated proteinlike 1 (GABARAP-L1). In living cells N63 Atg4D stimulates the delipidation of GABARAP-L1 whereas siRNA silencing of the gene expressing Atg4D abrogates GABARAP-L1 autophagosome formation and sensitises cells to starvation and staurosporine-induced cell death. Interestingly Atg4D overexpression induces apoptosis which is preceded by the caspase-independent recruitment of Atg4D to mitochondria and is facilitated by a putative C-terminal Bcl-2 homology 3 (BH3) domain. Atg4D also acquires affinity for damaged mitochondria in cells treated with hydrogen peroxide. These data suggest that Atg4D is an autophagy regulator that links mitochondrial dysfunction with apoptosis.

Keywords:
Autophagy; Autophagin; Atg4; Atg8; LC3; GABARAP-L1

Materials & Methods:
Fluorescence microscopy live-cell imaging and autophagosome quantitation Wide-field fluorescence images were obtained using an Olympus IX-71 inverted microscope (60 Uplan Fluorite objective 0.65-1.25 NA at maximum aperture) fitted with a CoolSNAP HQ CCD camera (Photometrics Tucson AZ) driven by MetaMorph software (Molecular Devices). Confocal images were obtained using a Leica AOBS SP2 microscope (63 PLAPO objective 1.4 NA) at 0.2 μm z-steps. For immunofluorescence cells were fixed in 2% formaldehyde (methanol-free EM grade; TAAB Aldermaston UK) followed by permeabilisation with 0.1% Triton X-100 or in –20°C methanol. Cells were routinely stained with DAPI and mounted in Mowiol containing 25 mg/ml DABCO anti-fade. Live-cell imaging was carried out using the Olympus IX-71 system. Halogen lamp illumination was used for both transmitted light and for epifluorescence to extend cell viability (Moss et al. 2006). Cells were maintained in CO2-independent DMEM (Invitrogen Paisley UK) at 37°C in 3-cm cell imaging dishes (MatTek Ashland MA). Automated autophagosome scoring was carried out using the Top Hat morphology filter in MetaMorph software selecting for round objects of diameter 5 pixels (=1 μm) or less.

Microscopic Technique
Confocal Microscopy, time-lapse, Fluorescence Microscopy

Cell Type(s)
HeLa