Validation of two potential mitochondrial fission factors identified by a genome wide high content imaging screening

Mitochondrial dynamics, consisting in fission and fusion events, are essential for cellular health. Dysregulation of these processes has been associated with various human disorders. In this study, we investigated the roles of two proteins, CSPα (Cystein string Protein α) and DTNA (Dystrobrevin α), in mitochondrial fission. Using Opa1 knockout (KO) Mouse Embryonic Fibroblasts (MEFs), we silenced CSPα and DTNA and observed altered mitochondrial morphologies. Loss of CSPα and DTNA led to increased elongated mitochondria, suggesting their involvement in promoting fission. Additionally, experiments in Mfn1 knockout MEFs corroborated the results obtained, confirming the roles of CSPα and DTNA in mitochondrial morphology modulation. To confirm their involvement in mitochondrial fission, and address challenges in distinguishing between naturally elongated mitochondria and siRNA-induced elongation, we used FCCP, to trigger extensive basal mitochondrial fission. FCCP-treated cells revealed distinct differences between control (siNT) and other siRNA-transfected cells (siCSPα, siDTNA, and siDrp1), emphasizing the specific impact of CSPα and DTNA silencing. These results confirmed that our 2 candidates seems involved in mitochondrial fission regulation. Our study provides crucial insights into the regulatory mechanisms governing mitochondrial dynamics, enhancing our understanding of these processes in cellular physiology and disease pathology.