Fig. 3. Mating structure adhesion is required for HAP2 trimer formation.

a Ciliary ectosomes induced gamete activation, but failed to initiate HAP2 trimer formation. HAP2-HA minus gametes were mixed with ciliary ectosomes for 30 min and assessed for gamete activation, as indicated by a cell wall loss assay (upper panel), and for formation of SDS-resistant trimers (lower panel). The asterisk indicates the trimer band detected upon mixing HAP2-HA minus gametes with WT plus gametes for 10 min. b Cell wall loss in minus gametes mixed for 10 min with fus1 plus gametes or with WT plus gametes. Results for cell wall loss assays in a and b are averages of 5 independent experiments with samples of 3 ×10⁷ cells assessed in each. c Phase-contrast micrographic images from a video (Supplementary Movie 1) of an interacting fus1 plus gamete and a WT minus gamete. The mating structure-bearing apical regions of cells collided, but failed to adhere during the pushing and pulling that occur during ciliary adhesion. Scale bars, 5 μm. n ≥ 100. d fus1 plus gametes fail to form trimers during gamete interactions. fus1 plus gametes or WT plus gametes were mixed with HAP2-HA minus gametes for 10 min and subjected to semi-native SDS-PAGE and immunoblotting. The alpha-tubulin loading controls are shown on lower blots in both a and d. The blot images in a and d are representative of 3 independent experiments.