Nicholas R Ader1,2, Patrick C Hoffmann1, Richard J Youle 2, Wanda Kukulski1* 1Cell Biology Division, MRC Lab of Mol. Bio., Cambridge, UK; 2Biochemistry Section, Surgical Neurology Branch, NINDS, NIH, Bethesda, MD *Correspondence: firstname.lastname@example.org
We investigate mitochondrial membrane restructuring mediated by the protein Bax during apoptosis. This form of cell death is made irreversible by the release of cytochrome c from the mitochondria into the cytosol, a process facilitated by Bax. A pore-like model for Bax-mediated cytochrome c release has been suggested, but direct evidence is sparse for how this pore forms and for its in vivo appearance. First, we applied correlative light microscopy and electron tomography (ET) to resin-embedded cells following either GFP-Bax overexpression or drug-induced apoptosis. These data revealed variably sized ruptures in the outer mitochondrial membrane near the correlated localization of GFP-Bax. In the intermembrane space of ruptured mitochondria, we often observed ribosomes. Their presence in a normally ribosome-free compartment offered both secondary confirmation of an open outer membrane and evidence of an influx of cytosol into the intermembrane space. In addition, we found that Bax activity leads to major restructuring of the inner membrane. We observed multiple matrices in one mitochondrion, apparently the result of asymmetric fission of the inner membrane. Some matrices appeared to be swollen, dilute, and devoid of cristae. Next, we used cryo ET to image cryo focused ion beam milled cells to obtain insights on Bax-induced changes to the molecular architecture of mitochondria preserved in a near-native state. These data provided high-resolution information on the outer membrane ruptures. Furthermore, we observed a redistribution of ATP synthases from cristae to the boundary membrane, indicating flattening of the cristae. We also identified mitochondrial matrices that have fully shed their outer membrane. Finally, strong GFP-Bax signals correlated to regions of ribosome exclusion in the cytosol. These exclusion regions are seen in both resin-embedded and cryo ET data, consistent with previous reports of large Bax clusters. In summary, we find that Bax activity during apoptosis causes ultrastructural as well as molecular restructuring of mitochondria. These changes may be important for cytochrome c release and/or downstream apoptotic signaling events.
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