The localization and function of myomaker during myoblast fusion. Multinucleated skeletal muscle fibers form through the fusion of myoblasts during development and regeneration. Previous studies identified myomaker (Tmem8c) as a muscle-specific membrane protein essential for fusion (1). In our recent study (collaboration with Dr. Doug Millay, Cincinnati Childrens Hospital Medical Center) we explored the localization of endogenous myomaker in muscle cells (2). We found myomaker at the plasma membrane and also in the Golgi and post-Golgi vesicles. Trafficking of myomaker is regulated by palmitoylation of C-terminal cysteine residues that allows Golgi localization. To examine whether myomaker functions at the myoblast fusion stage or only prepares the cells for fusion we applied a synchronized fusion approach developed in earlier studies of our lab (1). We reversibly blocked fusion of myoblasts without blocking pre-fusion stages using lysophosphatidylcholine (LPC), an inhibitor of membrane merger. We accumulated ready-to-fuse cells in the presence of LPC and then removed LPC to allow fusion. Myomaker antibody applied at the time of LPC removal blocked lipid mixing and content mixing indicating that Myomaker at the plasma membrane at the time and place of fusion is directly involved in the early stages of myoblast fusion. Phosphatidylserine at the surface of osteoclast precursors regulates their fusion. Bone-resorbing multinucleated osteoclasts play central role in the maintenance and repair of our bones. In our recent study (3) we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. As for myoblast fusion (4) and in our earlier work on osteoclast fusion (5), we accumulated ready-to-fuse cells in the presence of LPC and then removed it to study synchronized cell fusion. We found osteoclast fusion to require the DC-STAMP- dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular phosphatidylserine-binding proteins annexins (annexin A5 in human osteoclasts), which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in C. elegans, cell fusion step in osteoclastogenesis is controlled by a phosphatidylserine coordinated activity of several proteins. In addition to identification of protein and lipid players in osteoclast fusion, in our recent studies we have suggested new ways of unbiased presentation of cell fusion at given conditions that combine empirical cumulative distribution function for the sizes of multinucleated cells with the total number of cell-cell fusion events, which generate these cells (3,6). References: 1. Millay DP, O'Rourke JR, Sutherland LB, Bezprozvannaya S, Shelton JM, Bassel-Duby R, et al. Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature 2013;499:301-5 2. Gamage DG, Leikina E, Quinn ME, Ratinov A, Chernomordik LV, Millay DP. Insights into the localization and function of myomaker during myoblast fusion. J Biol Chem 2017;292:17272-89 3. Verma SK, Leikina E, Melikov K, Gebert C, Kram V, Young MF, et al. Cell-surface phosphatidylserine regulates osteoclast precursor fusion. J Biol Chem 2018;293:254-70 4. Leikina E, Melikov K, Sanyal S, Verma SK, Eun B, Gebert C, et al. Extracellular annexins and dynamin are important for sequential steps in myoblast fusion. J Cell Biol 2013;200:109-23 5. Verma SK, Leikina E, Melikov K, Chernomordik LV. Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin. Biochem J 2014;464:293-300 6. Verma SK, Chernomordik LV, Melikov K. An improved metrics for osteoclast multinucleation. Sci Rep 2018;8:1768

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Budget End
Support Year
27
Fiscal Year
2018
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U.S. National Inst/Child Hlth/Human Dev
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Verma, Santosh K; Chernomordik, Leonid V; Melikov, Kamran (2018) An improved metrics for osteoclast multinucleation. Sci Rep 8:1768
Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran et al. (2018) Cell-surface phosphatidylserine regulates osteoclast precursor fusion. J Biol Chem 293:254-270
Zaitseva, Elena; Zaitsev, Eugene; Melikov, Kamran et al. (2017) Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine. Cell Host Microbe 22:99-110.e7
Gamage, Dilani G; Leikina, Eugenia; Quinn, Malgorzata E et al. (2017) Insights into the localization and function of myomaker during myoblast fusion. J Biol Chem 292:17272-17289
Valansi, Clari; Moi, David; Leikina, Evgenia et al. (2017) Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens. J Cell Biol 216:571-581
Zicari, Sonia; Arakelyan, Anush; Fitzgerald, Wendy et al. (2016) Evaluation of the maturation of individual Dengue virions with flow virometry. Virology 488:20-7
Kozlov, Michael M; Chernomordik, Leonid V (2015) Membrane tension and membrane fusion. Curr Opin Struct Biol 33:61-7
Chernomordik, Leonid V; Kozlov, Michael M (2015) Myoblast fusion: playing hard to get. Dev Cell 32:529-30
Leikina, Evgenia; Defour, Aurelia; Melikov, Kamran et al. (2015) Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles. Sci Rep 5:18246
Uygur, Berna; Abramo, Katrina; Leikina, Evgenia et al. (2015) SLUG is a direct transcriptional repressor of PTEN tumor suppressor. Prostate 75:907-16

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