MCL1 is a member of the anti-apoptotic BCL2 family of proteins that exert their anti-apoptotic function at least in large measure by sequestering BH3-domain-containing pro-apoptotic proteins within the outer mitochondrial membrane. Deletion of the transmembrane (TM) domain of MCL1 abolished the anti-apoptotic function of MCL1.
In this study we expressed various isoforms of MCL1, which were shortened and had modifications or deletions of the TM domain, in an expression vector to monitor the effects each isoform had on MCL1’s subcellular translocation. Using florescent reporters on the mitochondria and MCL1 we are able to compare the effects of the various isoforms and characterize the role of the TM domain using fluorescence microscopy. It was found that MCL1 TM has different localization patterns than the wild type. MCL1’s short, pro-apoptotic splicing form also shows different localization than the wild type, with MCL1 being located throughout the cytosol.
With over 200 point mutations of MCL1 created by the Golub lab, the assay outlined in this project of tagging MCL1 and being able to monitor expression would allow us to test MCL1’s translocation based on each of these point mutant forms, as well as allow us to screen MCL1 in the presence of small molecules to discover the small molecules’ effects on MCL1’s translocation. These could give us clues to how these genes and molecules affect MCL1 and how they could possibly be targeted in cancerous cells.
PROJECT: Defining elements required for MCL1 translocation
My summer in the SRPG program truly changed my life. I feel more confident, more prepared, and more excited about my own career in science. After this experience I feel like I’ve gained another family with my Broad colleagues, my SRPG program directors, and especially with my fellow SRPG students.