Myomaker and Myomerger Work Independently to Control Distinct Steps of Membrane Remodeling during Myoblast Fusion

Abstract

Classic mechanisms for membrane fusion involve transmembrane proteins that assemble into complexes and dynamically alter their conformation to bend membranes, leading to mixing of membrane lipids (hemifusion) and fusion pore formation. Myomaker and Myomerger govern myoblast fusion and muscle formation but are structurally divergent from traditional fusogenic proteins. Here, we show that Myomaker and Myomerger independently mediate distinct steps in the fusion pathway, where Myomaker is involved in membrane hemifusion and Myomerger is necessary for fusion pore formation. Mechanistically, we demonstrate that Myomerger is required on the cell surface where its ectodomains stress membranes. Moreover, we show that Myomerger drives fusion completion in a heterologous system independent of Myomaker and that a Myomaker-Myomerger physical interaction is not required for function. Collectively, our data identify a stepwise cell fusion mechanism in myoblasts where different proteins are delegated to perform unique membrane functions essential for membrane coalescence.

Keywords: Myomaker; Myomerger/Minion/Myomixer; cell-cell fusion; membrane fusion; muscle development.

Figure 1.. Distinct roles for Myomaker and Myomerger during the fusion process.

(A) Unsynchronized cell fusion assay (no LPC) to monitor lipid and content mixing. Cells are differentiated for 48 hours then one population is labeled with both lipid (red, DiI) and content (green cell tracker) probes and mixed with unlabeled cells. Hemifusion is identified as mononucleated cells with only the red lipid probe whereas complete fusion is identified as multinucleated cells with both probes, and these indices were scored 24 hours after mixing the labeled and unlabeled cells. (B) Immunofluorescence images of WT, Myomaker^−/− and Myomerger^−/− C2C12 myoblasts after lipid and content mixing. Arrows indicate hemifused cells, whereas arrowheads mark complete fusion. (C) Quantification of hemifusion (left, n=3 independent experiments) and complete fusion (right, n=3 independent experiments) as a percentage to total cells. (D) Schematic of content mixing to assess formation of fusion pores. (E) Fusion pore formation was identified by the appearance of double labeled cells (arrows) in WT and Myomerger^−/− C2C12 myoblasts after 24 hours. (F) Quantification of (E) (n=3 independent experiments) as a percentage of total cells. Statistical analyses and data presentation: (C) (Hemifusion), two tailed Student’s t-test; (C) (complete fusion), (F), Mann-Whitney test; box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 50 μm. See also Figure S1.

Figure 2.. Compensation of Myomerger deficiency by heterologous membrane-stressing treatments.

(A) Schematic illustrating hypotonic shock fusion rescue experiment. Complete fusion is indicated by the presence of multinucleated cells labeled with both lipid and content probes. (B) Representative images of WT, Myomaker^−/− and Myomerger^−/− C2C12 cells differentiated for 48 hours, labeled with appropriate probes and plated in differentiation medium for 24 hours, then treated with PBS:H₂O (1:3) (hypotonic shock) for 60 seconds. (C) Quantification of complete fusion (n=3 independent experiments) as a percentage of cells of the same genotype that did not receive hypotonic shock. Myomerger^−/− C2C12 myoblasts showed a significant increase in fusion when treated with hypotonic shock. (D) Treatment with Octyl-β-Glucoside (OG) on day 3 of differentiation (Diff.) induced fusion in Myomerger^−/− myoblasts (arrows) but not in Myomaker^−/− C2C12 myoblasts. (E) Treatment with Magainin 2 (Mag2) for 20 hours on day 3 of differentiation induced myotube formation in Myomerger^−/− C2C12 myoblasts (arrows) but had no effect on Myomaker^−/− cells. For (D) and (E) the cells were immunostained with myosin antibodies to identify differentiated muscle cells and Hoechst dye was used as a nuclear stain. (F) The percentage of myosin+ cells that contain 3 or ≥4 nuclei after treatment with OG (n=3 independent experiments) or Mag2 (n=4 independent experiments) as an indicator of fusogenicity. Statistical analyses and data presentation: (C), (F), two tailed Student’s t-test; box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); ***P < 0.001, ****P < 0.0001. Scale bars, 50 μm. See also Figure S2.

Figure 3.. Myomaker does not require Myomerger to efficiently initiate hemifusion structures.

(A) Myoblasts were labeled with content probes (either orange cell tracker or green cell tracker) and differentiated for 48 hours. One set of myoblasts was allowed to fuse uninterrupted without LPC (Control (-LPC)), while others were treated with the synchronization agent LPC for 16 hours in differentiation medium to accumulate myoblasts that are ready-to-fuse. At the time of LPC removal, which allows cells to undergo hemifusion and complete fusion, the cells were exposed to either hypotonic shock (1:3 PBS:H₂O) or chlorpromazine (CPZ) for 60 seconds, washed and assayed for fusion after 30 minutes. (B) Representative images show fusion for myoblasts that were not exposed to LPC (Control (-LPC)) and myoblasts that were synchronized with LPC and then treated with CPZ. (C) Quantification of syncytium formation as the percentage of nuclei in multinucleated cells (defined as cells with 2 or more nuclei). Statistical analyses and data presentation: (C) Mann-Whitney test; box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); NS not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001; Scale bar, 50 μm.

Figure 4.. The C-terminal ectodomain of Myomerger functions from outside the cell.

(A) Immunoblot for Myomerger from the surface biotinylated fraction (IP: Streptavidin) and total lysates (input) from differentiated C2C12 myoblasts. Cells not treated with biotin were used as a control. Absence of the cytosolic protein (Tubulin) from the biotinylated fraction confirms surface-specific labeling. Data shown is representative from 3 independent experiments. (B) Schematic of the synchronized fusion assay. Myoblasts that had been differentiated for 48 hours were labeled with either DiI (lipid probe) or green cell tracker (content probe). Addition of LPC accumulates myoblasts that are ready-to-fuse and removal of LPC (LPC/Wash) allows cells to undergo hemifusion and complete fusion. Antibodies to Myomerger ectodomain, or IgG as a control, were added during this wash phase. (C) Quantification of synchronized fusion for WT C2C12 (n=3 independent experiments) myoblasts was achieved by assessing formation of mononucleated cells with both membrane and content probes (hemifusion, left) and multinucleated myotubes (complete fusion, right). (D) Incubation of rMBP-Myomerger^26-84 for 20 hours in differentiation media induces fusion of Myomerger^−/− (arrows), but not Myomaker^−/− C2C12 myoblasts. Cultures were immunostained with myosin antibodies to identify differentiated muscle cells and Hoechst dye was used as a nuclear stain. (E) The percentage of myosin⁺ cells that contain 3 or ≥4 nuclei after incubation with rMBP or rMBP-Myomerger^26-84 (n=4 independent experiments with two independent protein purifications) at various concentrations of recombinant protein. Statistical analyses and data presentation: (C), (E), Mann-Whitney test; box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001; Scale bar, 50 μm. See also Figures S3, S4, and S5.

Figure 5.. Myomerger functions to stress membranes.

(A) Schematic showing that disruption of the liposome membrane releases self-quenched Sulforhodamine B from the liposomes generating a fluorescence signal. (B) Liposome content release was monitored as an increase in Sulforhodamine B fluorescence and is presented as a percentage of the fluorescence measured after complete dequenching of the fluorescence by SDS application at the end of each experiment. Robust leakage was observed when liposomes were subjected to rMBP-Myomerger^26-84 but not to rMBP. (C) Analysis of leakage two minutes after the addition of various concentrations of recombinant protein (n=4 independent experiments with two independent protein purifications and two independent liposome preparations). (D) A permeabilization assay to determine an effect for Myomerger on the plasma membranes of cells. Myoblasts are incubated with water (induces osmotic swelling, membrane tension, and pore(s)) and fluorescent phalloidin (F-actin probe) for 30 seconds and detection of phalloidin⁺ cells indicates permeabilization. (E) Representative images show phalloidin labeling and Hoechst-labeled nuclei from proliferating WT C2C12 myoblasts and differentiating WT, Myomaker^−/−, and Myomerger^−/− C2C12 myoblasts after hypotonic shock. (F) Quantification of the percentage of phalloidin⁺ cells from E (n=3 independent experiments). Statistical analyses and data presentation: (C) two-tailed Student’s t-test; (F) Mann-Whitney test; (C) mean ± standard deviation; (F) box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001; Scale bar, 50 μm. See also Figure S5.

Figure 6.. Myomerger drives fusion completion independent of Myomaker.

(A) Schematic showing heterologous fusion assay. 3T3 fibroblasts expressing hemagglutinin (HA) were transfected with empty, Myomaker or Myomerger plasmids and mixed with erythrocytes that were labeled with both lipid and content probes. (B) At selected sub-optimal conditions, HA only drives hemifusion (fibroblasts labeled with the lipid probe, arrowhead). Since fibroblasts are much larger than erythrocytes, hemifusion events are seen as appearance of large cells labeled with only lipid probe. Expression of Myomerger but not Myomaker drives complete fusion (fibroblasts labeled with both lipid and content probes, arrows). (C) Quantification of hemifusion (left) and complete fusion (right) from (B) (n=3 independent experiments). Statistical analyses and data presentation: Mann-Whitney test; box-and-whisker plots show median (center line), 25th–75th percentiles (box) and minimum and maximum values (whiskers); *P < 0.05, ***P < 0.001, ****p < 0.0001. NS, not significant. Scale bar, 50 μm. See also Figures S6 and S7.

Figure 7.. Schematic of the division of fusogenic labor between Myomaker and Myomerger.

Myomaker and Myomerger independently control two distinct stages of fusion in myoblasts. Both proteins are likely expressed simultaneously in differentiated fusion-committed myocytes. Colored proteins are shown during the stage at which they are essential although they may also be active during other times. At the first stage, Myomaker controls development of hemifusion competence and is indispensable for hemifusion connections (mixing of lipids (blue)), and Myomerger is not required for this step. The fusion reaction would stall at this stage without the presence of Myomerger, which at the second stage of the reaction generates membrane-stresses to drive the transition to complete fusion (green-yellow content mixing) in a Myomaker-independent manner. Note that the schematic is meant solely to illustrate how the independent activities of Myomaker and Myomerger cooperate during myoblast fusion, and the other factors that also impact the process are omitted for clarity.