Protein-mediated membrane remodeling

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Myoblast fusion, a key process in the development and regeneration of skeletal muscle, is one of the best characterized examples of cell-cell fusion. Two muscle-specific proteins have been discovered to be essential for myoblast fusion: Myomaker and Myomerger/Myomixer/Minion. In our earlier work we found that Myomaker is required for hemifusion, whereas the subsequent transition from hemifusion to complete fusion depends on the extracellularly localized region (ectodomain) of Myomerger. The role of Myomerger in myoblast fusion represents a paradigm applicable to all fusion events, the essence of which is the promotion of fusion pore formation in the hemifusion diaphragm (HD) that consists of the distal (inner) monolayers of the fusing membranes by proteins/protein regions that do not directly interact with these membrane monolayers. Considering that Myomerger is not involved in hemifusion but drives the transition from hemifusion to fusion, this protein presents an important model for analysis of the physical mechanisms underlying an, apparently, indirect effect of Myomerger and other proximal leaflet associated factors on the fusion pore formation. In our recent study to explain how Myomerger promotes hemi-to-full fusion transition in myoblast fusion, we hypothesized that Myomerger shifts the spontaneous curvature of the proximal membrane monolayers towards positive values. This shift generates additional tension in the distal monolayers composing the HD and, hence, promotes the fusion pore formation. We theoretically analyzed the effects of the positive spontaneous curvature of proximal membrane monolayers on the elastic stresses in HD and uncovered an HD rim mediated elastic crosstalk between the proximal membrane monolayers and the HD. The crosstalk elicits growing HD tension and fusion pore formation following increased proximal monolayer spontaneous curvature. We supported the suggested mechanism by experiments, in which we found that a synthetic polypeptide sMyomerger26-84 with an amino acid sequence corresponding to Myomerger protein lacking the N-terminal transmembrane domain of the protein (amino acids 125) generates positive spontaneous curvature of lipid monolayer. Furthermore, the fusion defect in Myomerger-deficient myoblasts can be partially rescued not only by application of sMyomerger26-84 but also by application of Lysophosphatidylcholine (LPC), a lipid of positive spontaneous curvature. A sufficiently strong shift of the spontaneous curvature in the proximal monolayers to positive values by LPC or by sMyomerger26-84 inhibited hemifusion and, consequently, fusion. However, the concentrations of sMyomerger26-84 and LPC that promoted hemifusion-to-fusion transition were considerably lower than those required for the hemifusion inhibition. We suggest that levels of Myomerger expression characteristic for fusion-committed myoblasts are in the range allowing Myomerger to promote rather than inhibit myoblast fusion. Our estimate for the surface density of sMyomerger26-84 that rescues fusion of Myomerger-deficient cells is comparable to the surface densities reported for viral fusogens. The physical mechanism, by which, according to our analysis, Myomerger drives fusion pore opening, can also underlie the effects of other proximal leaflet-associated factors on the fusion pore formation in diverse fusion processes.