Fig. s5

Recombinant Bouncer binding confirms the interaction between zebrafish Bouncer and the zebrafish trimeric complex, related to Figure 4 (A and B) PAE plots of the top 5 models of the sperm trimeric complex (Spaca6, Izumo1, and Tmem81) predicted to interact with the egg proteins Bouncer (in zebrafish) and JUNO (in mice and humans) (A), but not predicted to interact with the sperm protein SPACA4 in mice (B). Units: amino acid residues; color bar: expected position error in angstroms. (C) Construct architecture of recombinant BIR2-Bouncer and chromatogram of BIR2-Bouncer size exclusion chromatography (SEC, blue trace). Dotted line traces a run of the same column resolving the gel filtration standard (BioRad). Arrow indicates the fraction analyzed by SDS-PAGE on the right (InstantBlue staining). SEC and PAGE reveal monomeric protein (calculated mass: 36.9 kDa). Experiments were performed using concentrated protein of the monomeric peak. (D) Dose-response curve of the fertilization capacity of wild-type sperm in in vitro fertilization (IVF) after incubation with recombinant Bouncer. To account for clutch-to-clutch variation, eggs were stripped from a single zebrafish female into two dishes. Sperm, which was either incubated with BIR2-Bouncer or control protein (BIR2 or BSA), was used to perform IVF. To determine the fertilization capacity, the fertilization rate of the Bouncer-incubated sperm was divided by the rate of the control. Each data point represents a split clutch from one female. Per tested concentration, at least three clutches were tested across six independent experiments. (E) To visualize surface expression of the sperm trimer in HeLa cells, the proteins were extracellularly tagged in the linker between the Ig-like domain and the transmembrane domain (TMD) with short epitope tags: Spaca6-2xV5-TMD, Izumo1-ALFA-TMD, and Tmem81-2xMyc-TMD. Left: counter-staining against the V5-, ALFA-, and myc-tags under non-permeabilizing conditions showed detectable levels of Spaca6 (magenta), Izumo1 (cyan), and Tmem81 (yellow). Even after accounting for bleed-through of the Izumo1 channel into the Tmem81 channel, a fraction of transfected cells carry detectable levels of all three proteins on the surface. Right: staining for Spaca6 and Izumo1 under permeabilizing conditions revealed that the vast majority of Spaca6 and Izumo1 proteins were retained within the cell. Images of permeabilized and non-permeabilized cells were acquired using identical imaging conditions; the pixel values of the non-permeabilized condition were multiplied by 20 to visualize the weaker signal. (F) Assessment of Bouncer binding to cells expressing either both Izumo1 and Spaca6 or only the single proteins. Top two rows: representative images of the Bouncer binding assay revealed that Bouncer binding requires both sperm factors. Bottom two rows: to visualize surface expression, extracellularly V5- or ALFA-tagged Spaca6 and Izumo1 were detected via counter-staining against V5 and ALFA under non-permeabilizing conditions, revealing detectable levels of Spaca6 (magenta) and Izumo1 (cyan) even when expressed individually. (G) Assessment of the surface expression of Spaca6 and Izumo1 between the tested constructs. Representative images of the surface staining of Spaca6 (magenta) and Izumo1 (cyan). Quantifications are shown in Figure 4D.