Cholesterol-Mediated Dimerization of Mitochondrial Translocator Protein TSPO: Super-Resolution Microscopy and Molecular Dynamics Insights

The translocator protein (TSPO) is a mitochondrial membrane protein that plays a critical role in cholesterol transport and steroid biosynthesis. While its functional significance is well established, the molecular mechanisms governing TSPO dimerization, particularly the regulatory role of cholesterol, remain incompletely understood. In this study, we first employed direct stochastic optical reconstruction microscopy (dSTORM) to experimentally investigate the organization and aggregation of TSPO in living cells under different cholesterol conditions. Our imaging results revealed that cholesterol stimulation led to the formation of smaller TSPO aggregates and an increase in the point density on the cell membrane, indicating that cholesterol alters the aggregation pattern of TSPO. To further interpret these findings at the molecular level, we conducted coarse-grained molecular dynamics simulations across varying cholesterol concentrations. The simulations demonstrated that cholesterol inhibits TSPO dimerization by reducing the number of stable dimers, likely due to modifications in the local membrane environment and direct interactions with TSPO. Although the dSTORM and molecular dynamics results show differences in aggregate size and oligomerization behavior, both approaches highlight the complex regulatory role of cholesterol in TSPO organization. By integration of high-resolution imaging with computational modeling, this study provides new insights into the cholesterol-mediated modulation of TSPO dimerization, with implications for mitochondrial function and cholesterol homeostasis.