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Mitochondrial membrane contact sites

Periodic Reporting for period 1 - MITOCHONTACTS (Mitochondrial membrane contact sites)

Reporting period: 2016-05-01 to 2018-04-30

Our body works like a commune. The multitude of tasks our body has to meet are performed in coordination between distinct organs such as the heart, the liver and the brain. This principle of division of labor goes down to the smallest living unit in our body, the cell. We have 50 trillion cells in our bodies and they each perform many functions. Different functions that a cell has to fulfill are carried out by intracellular structures termed organelles. For example, mitochondria function as the power plants of the cell, and lipid droplets are energy storehouses. The inside of each organelle is protected by a surrounding membrane.

The presence of organelles has numerous benefits for the cell. They offer separate cellular reaction chambers and thus allow for the cell to perform diverse functions at the same time, even functions that are conflicting with each other or that require different environmental conditions. On the down-side however, maintaining organelles also comes at a cost. For example, organelles require an elaborate communication system in order for the cell to function in coordination. One key way of organelle communication is via contact sites, places where two organelles are physically linked to each other via special proteins that are spanning the membranes of both organelles.
They key objective of this project was to gain a deeper understanding about the molecules underlying formation of contact sites, their functional roles, and their relevance for the cell.
The main technology used in this project were robotic, unbiased searches for novel players in contact site biology using microscopy. Using this technique, we discovered a novel machinery that we termed lipid droplet organization (LDO) machinery. The LDO machinery consists of at least two different proteins, Ldo45 and Ldo16, that sit on just a subset of cellular lipid droplets. Due to the presence of LDO proteins, these lipid droplets acquire special features as compared to other lipid droplets. Ldo16 mediates positioning of these lipid droplets in a special place in the cell, forming contact sites with two other organelles. Ldo45 on the other hand brings a defined set of proteins to these unique lipid droplets. Our results suggest a direct link between formation of contact sites and organelle specialization, indicating a novel role for contact sites (Eisenberg-Bord et al. & Bohnert, JCB 2018).

A further key technology in this project was the creation of special constructs that we termed contact site reporters, which allow the visualization of contact sites using fluorescence microscopy. These reporters contributed to the description of the full molecular structure responsible for formation of contact sites between mitochondria and the vacuole/lysosome, an organelle required for degradation and storage of diverse types of molecules (González Montoro et al., 2018).

Contact sites only moved into the focus of research within the last decade. Therefore, many basic questions are still open, and we contributed to the conceptual developments in the field with three review articles. The key players in contact sites are proteins termed tethers, which physically bridge the membranes of the organelles forming the contact. The importance of tethers is generally accepted, but a clear definition of what a tether is had been missing. We compared different types of contact sites and extracted a list of minimal requirements, including experimental confirmation of (1) a defined location in the area where two organelles are attached to each other; (2) the ability of the tether to tightly bind to the membranes of both organelles, and (3) the confirmation of a tethering force exerted by the machinery in question (Eisenberg-Bord et al. & Bohnert, Dev Cell 2016). In a subsequent work, we compared the features of classical contact sites as established by membrane enclosed organelles to contact sites formed by lipid droplets, which have an atypical surface. Indeed, lipid droplet contact sites have unique features. Next to tether proteins, lipid droplet contact sites can contain special organelle connections made from lipids that are never found in classical contact sites, and that might impact contact site functions (Schuldiner & Bohnert, BBA 2017). Finally, we contributed a comment article aiming at outlining future directions in the field (Bohnert & Schuldiner, Nat Rev Mol Cell Biol 2018).

Throughout its funding period, the project received pronounced visibility through oral presentations in international meetings, among them the FASEB SRC “Lipid droplets: Dynamic organelles in metabolism and beyond”, Snowmass, USA; the 14th international congress on yeasts (ICY14), Awaji Island, Japan; the 28th international conference on yeast genetics and molecular biology (ICYGMB), Prague, Czech Republic; the Symposium “Cellular microcompartments: From physiology to their analytics” (CRC 944), Osnabrück, Germany; as well as the CRC 1190 seminar series, Göttingen, Germany.
"The project has promoted the progress in contact site research both by contributing to the conceptual developments in the field, and through discovery of previously unknown molecular players in organelle communication.
Most importantly, discovery of the LDO machinery has opened a new area of research. Specialization of subpopulations within a cellular pool of organelles has been observed in the past, but the molecular mechanisms underlying this phenomenon have been unclear. The LDO machinery is a key player in this process, both mediating targeting of special proteins to a lipid droplet subpopulation, and at the same time dictating positioning of these organelles adjacent to partner organelles. These findings indicate that there might be a direct link between formation of contact sites on one hand and functional organelle specialization on the other hand, a mechanism that might apply to further contact sites in the cell. Importantly, we found a physical and functional link between the LDO machinery and Seipin, a lipid droplet biogenesis factor that has a key role in the human disease Berardinelli-Seip congenital generalized lipodystrophy and in neurological seipinopathies (Eisenberg-Bord et al. & Bohnert, JCB 2018).

Publications:
1.) Eisenberg-Bord, M., Shai, N., Schuldiner, M.#, and Bohnert, M.# (2016). A tether is a tether is a tether: Tethering at membrane contact sites. Dev. Cell 39, 395-409.
#corresponding
2.) Schuldiner, M., and Bohnert, M.# (2017). A different kind of love – lipid droplet contact sites. Biochim. Biophys. Acta 1862, 1188-1196.
#corresponding (selected for cover)
3.) Eisenberg-Bord, M., Mari, M., Weill, U., Rosenfeld-Gur, E., Moldavski, O., Castro, I.G. Soni, K.G. Harpaz, N., Levine, T.P. Futerman, A.H. Reggiori, F., Bankaitis, V.A. Schuldiner, M.#, and Bohnert, M.# (2018). Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation. J. Cell Biol. 217, 269-282.
#corresponding
4.) González Montoro, A., Auffahrt, K., Hönscher, C., Bohnert, M., Becker, T., Warscheid, B., Reggiori, F., van der Laan, M., Fröhlich, F., and Ungermann, C. (2018). Vps39 interacts with Tom40 to establish one of two functionally distinct vacuole-mitochondria contact sites. Dev. Cell 45, 621-636.
5.) Bohnert, M.#, and Schuldiner, M.# (2018). Stepping outside the comfort zone of membrane contact site research. Nat. Rev. Mol. Cell Biol. doi: 10.1038/s41580-018-0022-1
#corresponding
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At contact sites, organelles communicate through close physical links.