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From Isolated Compartments to Intracellular Networks: Deciphering Interorganelle Communication

Final Report Summary - ORGANET (From Isolated Compartments to Intracellular Networks: Deciphering Interorganelle Communication)

Organelles exchange metabolites and information to coordinate essential aspects of cellular physiology, thus behaving as an integrated intracellular network. For example, the endoplasmic reticulum (ER) makes contacts with several organelles, including the plasma membrane, mitochondria, the Golgi apparatus, and lysosomes. These contacts allow the coordination of calcium and lipid homeostasis across the cell. Yet, little is known about their molecular nature. A long-standing question in cell biology has been how membrane-rich mitochondria receive lipids synthesized in the ER in the absence of vesicular transport between the two organelles. One model suggests that lipids directly shuttle from one organelle to the other at sites of contact, although the hypothesis lacked molecular details.

During this research period we tackled 1) the architecture of ER-mitochondria contacts in yeast, 2) the function of these contact sites, 3) their regulation, and 4) their conservation in higher eukaryotes.
We have discovered that ER-mitochondria Encounter Structures in yeast are made of lipid transporting proteins, enlightening both the architecture and function of these contact sites. The lipid-exchange function was, however, not straightforward to demonstrate since removing ERMES complex only marginally affected the lipid composition of mitochondria. We found that this was due to the presence of a redundant pathway involving an interaction between the vacuolar protein Vps13 and the mitochondrial protein Mcp1. Vps13 is conserved in mammals, and paralogs of Vps13 localize to ER-mitochondria contact, thus informing on the conservation of the machinery.
Moreover, while pursuing the only conserved subunit of the ERMES complex, Miro, we discovered a new mechanism by which mitochondria attach to growing microtubules to be distributed to daughter cells at the end of mitosis. This mechanism involves the interaction of Miro with CENP-F, a microtubule-binding protein that accumulates along the cell cycle. However, probing the significance of this interaction by generating mouse models in which this interaction was abrogated failed at revealing a function.

The regulation of contact site was an unknown field. It was known, however, that ER-mitochondria contacts regulated mitochondrial dynamics and in particular, mitochondrial fission. We hypothesized that this might be an effect of mechanical forces applied on mitochondria from the encounter of the two organelles. We tested this by developing methodologies to apply force on mitochondria, and found that such a stimulus, at physiological level, was sufficient to trigger fission. We found moreover a suitable molecular mechanism to explain the phenomenon; the protein MFF, showing a preference for mitochondria of smaller diameter, accumulates on mechanical constrictions and recruits the fission machinery. Thus, organelle contacts that incur force onto mitochondria can be resolved through the division of the latter, thus regulating the strength of force applied to the mitochondrial network.

Finally, prompted by the discovery of redundant lipid exchange routes, we identified a need for a genetic screening method to uncover gene function in spite of the presence of redundant pathways. We therefore developed the SAturated Transposon Analysis in Yeast (SATAY), a method that allows screening multiple yeast strains in multiple media to identify genes that are required in given sets of genetic or environmental conditions. This method is now being widely adopted by the S. cerevisiae community and promises to become the method of choice for yeast genomics.