Exploiting Chemical Modulation of Mitochondrial Dynamics during Melanogenesis

Mitochondria participate in many intracellular processes: they are known for ATP production, but also for their roles in controlling lipid metabolism, calcium cation homeostasis, metabolism, and apoptosis. To handle all these functions, they are characterized by a particular structure that dynamically changes in response to cell needs. Interactions with other organelles play a role in adaptation to intracellular changes. In particular, the interaction between mitochondria and endoplasmic reticulum, called MERCs, has been recognized as a fundamental player in several signaling pathways, as Ca2+ transfer, phospholipid synthesis and exchange, mitochondrial fission and unfolded protein response (UPR). Consequently, MERCs impairment contributes to many diseases, such as neurodegenerative disorders, insulin resistance, and cancer. Considering this evidence, MERCs start to be considered as possible pharmacological targets. The molecules able to change the distance between the two organelles (chemical MERCs modulators) can exert their action through different mechanisms. Aim of this work was to identify some chemical MERCs modulators and define their mechanisms of action. We performed a chemical compound screen based on high content imaging to study mitochondrial dynamics. After validation, our approach revealed five molecules able to change MERCs independently from the mitochondrial morphology. In parallel, we developed a methodology for high throughput quantification of mitochondrial membrane potential in 2D and 3D models using the TMRM dye. By combining automated image analysis and machine learning we were able to distinguish melanoma cells from macrophages in co-culture, and to analyze the subpopulations separately. We also applied this approach to human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Then, we decided to apply our knowledge to a process whose molecular mechanisms are still unclear: melanogenesis, the process of melanin synthesis. Melanogenesis occurs in lysosome-related organelles called melanosomes, present within cells of the epidermis called melanocytes. Melanin is a biopolymer with pleiotropic roles in the cell, ranging from UV light photoprotection to ion chelation and ROS modulation. Considering melanogenesis as a protective cell response from stress factors, and the pivotal role of MERCs during the stress response, we hypothesized that the interplay between mitochondria and endoplasmic reticulum could contribute to melanogenesis as well. We observed that extrinsic factors, like UV, inducing melanogenesis impact also on mitochondrial dynamics. To study melanogenesis we also designed a high throughput approach to analyze several properties of melanosomes, such as their number, intensity and morphological parameters. In summary, this work contributes to the development of several methodologies for the study of mitochondrial biology (e.g., functionality, morphology, MERCs, interactions with melanosomes), but also new knowledge on the molecular pathways and on the pharmacology that regulate MERCs. Moreover, it also provides for the first time an analysis of the crosstalk between melanogenesis and mitochondria dynamics.