Protein-mediated membrane remodeling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Myoblast fusion, a key stage in the development and regeneration of skeletal muscle, is a multistep process starting from hemifusion controlled by muscle-specific protein Myomerger/Myomixer/Minion and followed by opening of a fusion pore controlled by another muscle-specific protein Myomaker. Molecular mechanisms by which Myomerger, a single-pass transmembrane protein containing 84-amino acids with an ectodomain that includes two -helices drives progression beyond early hemifusion events to complete fusion remain to be understood. In our earlier studies we demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. In our new work we explored specific contributions of different domains of Myomerger in functionally important protein-lipid bilayer interactions. We also examined how phosphatidylserine, a lipid previously implicated in diverse cell-cell fusion processes, including myoblast fusion, regulates Myomerger activity. Under fusion conditions, phosphatidylserine, normally present only in the inner leaflet of plasma membrane, is also transiently exposed in the outer membrane leaflet, where it interacts with Myomerger ectodomain. Complementing myoblast fusion assays and in vitro liposome assays, we found that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We characterized membrane insertion of different regions of Myomerger by evaluating changes in the spontaneous curvature of lipid monolayer. To characterize changes in this membrane property that is critically important for membrane fusion we used our recently published assay, in which we compare the effects of inserted molecules on the FRET efficiency between dyes attached to either lipid headgroups or lipid tails. We found that the membrane proximal, amphipathic Helix-1 is normally disordered and its -helical structure is induced by phosphatidylserine, facilitating interactions of this region with the membrane. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and promotes the membrane-stressing effects of Helix-1. The removal of either of the two -helices in Myomerger ectodomain dramatically reduced its effect on Frster resonance energy transfer, suggesting that both -helices are necessary for efficient insertion and curvature generation. Similarly, mutations in either of the -helices decreased insertion and positive curvature generation. Our findings reveal that Myomerger fusogenic activity is tightly-controlled event involving its two ectodomain helices, which are regulated by changes in the lipid composition of the fusing membranes, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final stage of myoblast fusion in formation of multinucleated muscle cells. A deeper understanding of the myoblast fusion mechanism could help develop new therapeutic strategies for genetic and acquired muscle diseases. The uncovered mechanism by which cells control the activity of proteins that merge membranes can underlie the fusion pore formation in diverse fusion processes and in other cell biological processes involving protein-induced membrane deformations.