Figure S3.
Fusion between BHK-JUNO to sperm and BHK-IZUMO1+VSV-G to oocyte fusion. (A and B) BHK cells transfected with pcDNA3.1-EGFP-MBD-nls alone (control) or together with pExpress1::JUNO were incubated with IZUMO1-mCherry sperm (magenta) and observed by fluorescence microscopy. Fusion was evaluated as the presence of EGFP-MBD-nls signal (green) inside the sperm heads (arrows) as opposed to bound cells (arrowheads). 175 out of 900 cells expressing JUNO presented at least one sperm fused to them (n = 3), while none of 900 control cells showed signs of fusion (n = 3). None of EGFP-MBD-NLS–positive (fused) sperm showed IZUMO1-mCherry signal, while 85% (n = 300) of EGFP-MBD-nls–negative (but bound) sperm carried IZUMO1-mCherry. The outline of the cells is represented with a dashed line. DAPI staining (blue). Scale bars, 20 µm. (C and D) BHK cells transfected with pCI::IZUMO1::H2B-RFP alone or together with a plasmid encoding for VSV-G were incubated with CD9-GFP oocytes (green) and observed by confocal microscopy. Fusion was evaluated as the presence of RFP-positive chromosomes (magenta) inside the cytoplasm of the eggs (arrows) as opposed to bound cells (arrowheads). For IZUMO1, no fusion was detected in >100 oocytes with cells attached, while for IZUMO1+VSV-G, 18 out of 45 oocytes with cells bound displayed fusion. To activate VSV-G activity, we exposed these oocytes to pH 2.5 for 30 s. The maximal intensity Z-projection, two different focal planes (Z-position 1 and 2), and DIC are shown. DAPI staining (blue). Scale bars, 20 µm. (E) Summary and working model. (a) Heterologous IZUMO1 expression in somatic cells in culture induces cell-to-cell fusion in a unilateral manner and independently of JUNO. The mutation W148A had no effect on the levels of content mixing compared to WT IZUMO1, but disrupted the binding of BHK cells to oocytes. The mutant FWW was still able to mediate BHK binding to the oocyte; however, it induced significantly lower levels of content mixing than WT IZUMO1. These results support the existence of two functional domains in IZUMO1, with binding to JUNO mediated through W148 on the hinge and cell-to-cell fusion dependent on three exposed aromatic residues on the four-helix bundle of the Izumo domain. (b) Working model for IZUMO1 activities. 1. Transient interaction between JUNO and IZUMO1 (“Binding”) mediated by IZUMO1 W148 on the hinge region (blue star). 2. Conformational change of IZUMO1, oligomerization (Inoue et al., 2015), and unilateral induction of gamete fusion (“Unilateral fusion”), enhanced by the action of F28, W88, and W113, the surface exposed aromatic residues on the four-helix bundle of IZUMO1 (pink stars). The action of secondary receptor(s) for IZUMO1 is also possible. IZUMO1 domain colors correspond to Fig. 4, A and C. 3. Following pore formation, there is cytoplasmic mixing.

Fusion between BHK-JUNO to sperm and BHK-IZUMO1+VSV-G to oocyte fusion. (A and B) BHK cells transfected with pcDNA3.1-EGFP-MBD-nls alone (control) or together with pExpress1::JUNO were incubated with IZUMO1-mCherry sperm (magenta) and observed by fluorescence microscopy. Fusion was evaluated as the presence of EGFP-MBD-nls signal (green) inside the sperm heads (arrows) as opposed to bound cells (arrowheads). 175 out of 900 cells expressing JUNO presented at least one sperm fused to them (n = 3), while none of 900 control cells showed signs of fusion (n = 3). None of EGFP-MBD-NLS–positive (fused) sperm showed IZUMO1-mCherry signal, while 85% (n = 300) of EGFP-MBD-nls–negative (but bound) sperm carried IZUMO1-mCherry. The outline of the cells is represented with a dashed line. DAPI staining (blue). Scale bars, 20 µm. (C and D) BHK cells transfected with pCI::IZUMO1::H2B-RFP alone or together with a plasmid encoding for VSV-G were incubated with CD9-GFP oocytes (green) and observed by confocal microscopy. Fusion was evaluated as the presence of RFP-positive chromosomes (magenta) inside the cytoplasm of the eggs (arrows) as opposed to bound cells (arrowheads). For IZUMO1, no fusion was detected in >100 oocytes with cells attached, while for IZUMO1+VSV-G, 18 out of 45 oocytes with cells bound displayed fusion. To activate VSV-G activity, we exposed these oocytes to pH 2.5 for 30 s. The maximal intensity Z-projection, two different focal planes (Z-position 1 and 2), and DIC are shown. DAPI staining (blue). Scale bars, 20 µm. (E) Summary and working model. (a) Heterologous IZUMO1 expression in somatic cells in culture induces cell-to-cell fusion in a unilateral manner and independently of JUNO. The mutation W148A had no effect on the levels of content mixing compared to WT IZUMO1, but disrupted the binding of BHK cells to oocytes. The mutant FWW was still able to mediate BHK binding to the oocyte; however, it induced significantly lower levels of content mixing than WT IZUMO1. These results support the existence of two functional domains in IZUMO1, with binding to JUNO mediated through W148 on the hinge and cell-to-cell fusion dependent on three exposed aromatic residues on the four-helix bundle of the Izumo domain. (b) Working model for IZUMO1 activities. 1. Transient interaction between JUNO and IZUMO1 (“Binding”) mediated by IZUMO1 W148 on the hinge region (blue star). 2. Conformational change of IZUMO1, oligomerization (Inoue et al., 2015), and unilateral induction of gamete fusion (“Unilateral fusion”), enhanced by the action of F28, W88, and W113, the surface exposed aromatic residues on the four-helix bundle of IZUMO1 (pink stars). The action of secondary receptor(s) for IZUMO1 is also possible. IZUMO1 domain colors correspond to Fig. 4, A and C. 3. Following pore formation, there is cytoplasmic mixing.

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