Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-XL, correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H 2 O 2 (200 mM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF b1-or H 2 O 2 -induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.

Bcl-x(L) is a potent inhibitor of apoptosis. While Bcl-x(L) can be bound to mitochondria, a substantial fraction, depending on the cell type or tissue, is found in the cytosol of healthy cells. Gel filtration and crosslinking experiments reveal that, unlike monomeric Bax, Bcl-x(L) migrates in a complex of approximately 50 kDa in the cytosol. Co-immunoprecipitation experiments indicate that Bcl-x(L) in the cytosol forms homodimers. The C-terminal hydrophobic tails of two Bcl-x(L) molecules are involved in homodimer formation, and analysis of mutants demonstrates that the C-terminal lysine residue and the G138 residue lining the BH3-binding pocket are required for homodimerization. The flexible loop preceding the C-terminal tail in Bcl-x(L) is longer than that of several monomeric Bcl-2 family members and is a requisite for the homodimer formation. Bad binding to Bcl-x(L) dissociates the homodimers and triggers Bcl-x(L) binding to mitochondrial membranes. The C-terminal tail of Bcl-x(L) is also required to mediate Bcl-x(L)/Bax heterodimer formation. Both mitochondrial import and antiapoptotic activity of different Bcl-x(L) mutants correlate with their ability to form homodimers.

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