Mitofusin Disease Variant Provides Insight into Mitochondrial Tethering and Fusion

Abstract

Mitochondria are dynamic organelles that produce the energy we need to move, grow, and think. Every cell, from muscles in the heart to neurons in the brain, has a unique and ever-changing requirement for energy. In order to adapt to the needs of each cell, mitochondria adjust shape through fusion, division and movement. Mitochondrial fusion in particular requires two molecular machines, called mitofusins. These mitofusins, Mfn1 and Mfn2, cooperate to drive membranes together through a mechanism that couples GTP hydrolysis, oligomerization, and conformational changes. Despite the importance of mitochondrial fusion in cell health, much of this mechanism is still unknown. In this work, I utilize a disease associated variant, Mfn2S350P and the equivalent Mfn1S329P variant in order to perform a structure-function analysis. I found that expression of either variant led to a perinuclear clustering of mitochondria, which differs from the reticular morphology of healthy cells. Mitochondria within the cluster do not have a connected mitochondrial matrix, indicating that individual mitochondria are in close proximity as opposed to fusing into one large mitochondria in the perinuclear space. Interestingly, microtubule-based transport of the mitochondria to the perinuclear space is not responsible for perinuclear clustering. Instead, clusters appeared to be caused by interactions between mitofusins across mitochondrial membranes, forming the mitofusin ¬trans complex. This indicates mitochondria are tethered together, but unable to proceed with full fusion. I also found that GTP hydrolysis is necessary for mitochondrial cluster formation, indicating that GTP hydrolysis is necessary for the formation of the trans complex. Additionally, I made progress in developing a method to measure conformational changes in full length mitofusin on the surface of isolated mitochondria based on the recently published technique, ACCuRET. Taken together, the work presented in my dissertation reveals unique insight into mitochondrial tethering and fusion and makes progress toward techniques to further analyze the molecular mechanism of mitofusin.