c-Src Modulates Endoplasmic Reticulum-Mitochondria Distance

Introduction: Physical interactions of the endoplasmic reticulum (ER) and mitochondria (MITO) are crucial for cellular functions, regulating the exchange of Ca 2+ , reactive oxygen species (ROS), and lipids. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, serves as one of the key ER-MITO tethering structures. We recently reported that in primary cardiac fibroblasts, increased activity of the proto-oncogene tyrosine-protein kinase Src (c-Src) leads to tyrosine phosphorylation of Mfn2 and a decreased distance between the ER and MITO, which correlates with enhanced mitochondrial Ca 2+ uptake, ROS generation, and cell proliferation. Hypothesis: c-Src modulates endoplasmic reticulum-mitochondria distance Objective: To test whether the inhibition of c-Src alters ER-MITO distance, mitochondrial Ca 2+ uptake, and ROS levels. Materials and Methods: To achieve cell compartment (i.e., ER-MITO contact site)-specific inhibition of c-Src, we utilized a dominant-negative c-Src protein (Src-DN) targeted to the outer mitochondrial membrane (OMM) by adding an OMM-targeting sequence (amino acid 1-33 of human TOM20) to the N-terminus of Src-DN (termed mt-Src-DN). HEK293T cells were transfected with Src-DN, mt-Src-DN, and their control vectors and subsequently used for immunoblotting and live cell imaging. Mitochondrial and cytosolic fractionations were performed using different centrifugation speeds with three independent experiments. Changes in ER-MITO distance were assessed through live cell imaging by measuring Förster resonance energy transfer (FRET) between an ER membrane-targeted monomeric cyan fluorescent protein (ER-CFP) and an OMM-targeted monomeric yellow fluorescent protein (MITO-YFP), respectively. Changes in mitochondrial Ca 2+ and cytosolic Ca 2+ were measured by mitochondria matrix-targeted Ca 2+ biosensor mt-Rchamp1h and Ca 2+ -sensitive dye Fluo-3. Results: Immunoblotting of mitochondrial and cytosolic fractionations confirmed that Src-DN was expressed and inhibited c-Src in both fractions. However, mt-Src-DN was preferentially expressed in mitochondrial fractions and inhibited c-Src activity only in mitochondrial fractions. Live-cell imaging revealed that the ER-MITO distance increased in the cells expressing mt-Src-DN compared to those transfected with the control vector (0.96±0.01 vs 1.00±0.02 in averaged FERT, n = 174 and 107, respectively). In mt-Src-DN-transfected cells, mitochondrial Ca 2+ uptake induced by cytosolic Ca 2+ elevation through ATP treatment (which stimulates endogenous G αq/11 protein-coupled P2Y receptor) was significantly slower compared to controls (30.9±4.1 vs 17.4±1.4 sec, n=47 and 37, respectively. p < 0.05). without significant changes in cytosolic Ca 2+ dynamics (n = 64 and 75, respectively). This suggests that the increased ER-MITO distance due to c-Src inhibition affects the mitochondrial Ca 2+ uptake profile. Lastly, the mitochondrial ROS levels were significantly lower in the mt-c-Src-DN group compared to the control groups, as assessed using the mitochondrial superoxide-sensitive dye Mitosox Red. Conclusion: c-Src located at the ER-MITO microdomains can modulate the distance between the ER and mitochondria via tyrosine phosphorylation of Mfn2. NIH/NHLBI R01HL171710 (to J.O.-U. and B.S.J.), R01HL160699 (to B.S.J.), and R01HL136757 (to J.O.-U.) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.