Strategies to Examine the Effect Of Bax and Bcl-xL on Individual Mitochondria

Programmed cell death is essential for development and tissue homeostasis. Bcl-2 family proteins, Bax and Bcl-xL, play relevant roles on intrinsic apoptotic pathways; they modulate mitochondrial dynamics and integrity. The physiological function of Bax and Bcl-xL in populations of cells has been addressed in the field, however; it is a mystery how they function in a population of mitochondria and whether activation of these proteins can lead to diverse cell death/survival outcomes. To address this, chimera constructs-YFP-BaxDC-Parkin, YFP-BclxLDC-Parkin were designed to target cell death/survival to a subset of mitochondria, assessing it through released cytochrome c. The hypothesis is that Bax recruited through Parkin would facilitate cell death and Bcl-xL would stall apoptosis. Parkin is recruited to compromised mitochondria. The H+ uncoupler CCCP was used to collapse membrane potential and recruit the chimeras. Our results indicated that recruitment of YFP-BaxDC-Parkin to the mitochondria induced more cytochrome c release than untreated cells. On the other hand, the YFP-BclxLDC-Parkin protected mitochondria, regardless of treatment. Unfortunately the lack of difference seen in Bcl-xL chimera, rendered it non-ideal for our studies. Therefore, an ongoing to tackle our question is using a FRB-FKBP (mTOR) domain-system to target Bax or Bcl-xL to DKO Mfn1/Mfn 2. This system allows more specific targeting to the mitochondria by using controlled rapamycin dosage. The DKO cells have a low basal membrane potential, which allows Parkin recruitment without CCCP treatment. Finally, studying the dynamics of the Bcl-2 proteins in mitochondria may provide insights into neurological disease, such as Parkinson’s disease.