ORGANELLE DYNAMICS AND MEMBRANE CONTACT SITES STUDY USING CRYO-SOFT X-RAY TOMOGRAPHY: THE ENDOPLASMIC RETICULUM AS A SCAFFOLD MEMBRANE FOR FISSION PROCESSES

Mitochondrial membrane contact sites (MCSs) play a crucial role in cellular function, yet there is still much to learn about MCSs dysfunction and its implications in human disease. GDAP1 and MFN2 are mitochondrial outer membrane proteins involved in membrane contacts in both between mitochondria, and mitochondria with diverse organelles. GDAP1 and MFN2 regulate mitochondria-endoplasmic reticulum (ER) contacts, the most well-characterized MCSs, but also participate in mitochondria-lysosome and mitochondria-peroxisome contacts. Interestingly, GDAP1 deficiency results in giant lysosomes and enlarged peroxisomes with abnormal distribution. Notably, the enzymatic capacity of these organelles remains unaffected, suggesting that GDAP1 dysfunction exclusively affects the membrane structure. MFN2 deficiency does not elicit this effect.
Pathogenic variants of the GDAP1 and MFN2 genes cause Charcot-Marie-Tooth (CMT) neuropathy, one of the most common inherited neurological disorders. Knowing in depth the precise role of GDAP1 and MFN2 in the MCSs could explain early events of this neuropathy.
Our research group has applied super-resolution fluorescence microscopy (computational superresolution and STED technologies) to analyze the morphology of mitochondria, lysosomes, and peroxisomes in GDAP1 and MFN2 deficient cells, as well as MCSs. Our current aim is to further investigate the inter-organelle connections with the ER, which has been proposed as a central hub for organelle dynamics. By employing cryo-soft X-ray tomography, we seek to elucidate the organellar landscape and membrane trafficking, thus uncovering the early events in the pathophysiology of CMT-related neuropathies and identifying potential therapeutic targets.