Dissecting House Wrecking and Housekeeping Roles of BAX in Mitochondria

Introduction: Multi-cellular organisms eliminate damaged cells as part of the normal cell life cycle by a regulated process of programmed cell death known as apoptosis. In mammals, apoptosis is regulated by the Bcl-2 gene family, which controls loss of outer mitochondrial membrane (OMM) permeability.  Two pro-apoptotic Bcl-2 members, Bax and Bak, oligomerize on the OMM forming Mitochondrial Apoptosis Induced Channel (MAC), from which death signals are released, representing the commitment step in apoptosis.  Bax and Bak’s role in the induction of apoptosis is well documented.  However, recent research suggests Bax and Bak may also play a housekeeping role in mitochondrial morphology.   Bax/Bak knockout cells show short, fragmented mitochondria when compared to wildtype cells. (Youle and Karbowski, 2011).  L63E cells (Bax/Bak knockouts where Bax is reintroduced with a BH3 domain mutation) result in exposure of the protein’s N-terminal end.  With this mutation, Bax still translocates to the mitochondria but fails to homo-oligomerize to form MAC (Kim and Cheng 2009). Objective: Determine whether the domain responsible for oligomerization of the Bax protein is also responsible for housekeeping role in mitochondrial morphology. Method: WT, DKO, and L63E mutants were plated on coverslips, grown in DMEM.  Coverslips were assembled in a Rose Chamber with Mitotracker and Hoerscht staining for the mitochondria and nuclei respectively and viewed under fluorescent microscopy.  ImageJ software was used to quantitatively compare mitochondrial morphology. Result: Preliminary examinations confirm Bax/Bak knockouts have substantially smaller mitochondria than wild types. Interestingly, re-introduction of mutant Bax seems to affect mitochondrial morphology. Further study is underway to determine to what extent morphology is restored and if other Bax domains are involved. Conclusion: Bax is emerging as a therapeutic candidate for neurodegeneration and cancer due to its role in apoptosis, but this study may give important insights into housekeeping roles that need to be considered.