This study identifies ether-linked phospholipids as modulators of Myomaker-mediated membrane fusion, revealing a lipid-centric perspective on the mechanisms driving myocyte fusion. Although we found no evidence of ceramidase activity for Myomaker, inhibiting sphingolipid biosynthesis enhanced fusion in both myocytes and BHK cells expressing Myomaker and Myomerger. These findings indicate that sphingolipids are not required for Myomaker function and may even act as antagonists of fusion. Lipidomic analyses under sphingolipid inhibition revealed an enrichment in ether lipids. Known for their fusogenic properties, these lipids were also enriched in Myomaker-containing lentiviral particles, indicating that membranes rendered fusion competent by Myomaker have higher concentrations of ether lipids. One possibility is that Myomaker may reside in, or help establish, lipid microdomains enriched in ether lipids. Functionally, increasing ether lipid levels, via Far1 overexpression or supplementation with the ether lipid precursor HG, was sufficient to induce Myomaker-dependent fusion even in the absence of Myomerger. Additionally, elevated ether lipid levels enhanced Myomaker’s localization to the plasma membrane and promoted externalization of PE and PS, hallmarks of membrane remodeling. Together, these findings suggest that ether lipids act as regulators of Myomaker activity and reveal a relationship between membrane lipid remodeling and Myomaker-mediated fusion.
Our work indicates that specific lipid classes, beyond their general fusogenic characteristics, can regulate protein-driven cell-cell fusion. One possible explanation for the ability of ether lipids to induce fusion in the presence of Myomaker is that they simply increase the amount of protein on the plasma membrane. While we detected an increase in plasma membrane-associated Myomaker after elevation of ether lipids, alternative ways to increase levels of Myomaker on the membrane, such as inhibition of autophagy, did not induce fusion, indicating that increases in plasma membrane Myomaker are not sufficient to induce fusion. This suggests that ether lipids influence the activity of Myomaker through additional mechanisms. One can hypothesize that an elevation in ether lipids promotes hemifusion-to-fusion transition by compensating for Myomerger’s activity.
