Fig. 2: DHC3 associates with SPMTs throughout ookinete morphogenesis.
A IFA of DHC3 expression during zygote to ookinete development. A schematic shows the developmental stages (I–V) of ookinete. Two parasite lines dhc3::6HA and 4Myc::dhc3 were analyzed. Three independent experiments. Scale bars: 5 μm. B Protein cortical distribution rate of DHC3 during ookinete development in (A). Values are means ± SD (n = 3 biological replicates). C IFA of DHC3 (HA) and SPMTs (α- and β-Tubulin) during ookinete development of the dhc3::6HA parasites. Three independent experiments. Scale bars: 5 μm. D Protein cortical distribution rate of DHC3 and Tubulin in (C). Values are means ± SD (n = 3 biological replicates). E Fractionation analysis of DHC3 in different stages (3, 6, and 12 h) of the dhc3::6HA ookinetes via immunoblot. Light fraction includes cytosolic proteins while heavy fraction includes membrane and cytoskeleton proteins. The cytosolic protein Enolase was used as a loading control. Two independent experiments. F Proximity ligation assay (PLA) detecting protein interaction between DHC3 and SPMTs in the dhc3::6HA ookinetes. Two independent experiments. Scale bars: 5 μm. G SPMT cytoskeleton association analysis of DHC3. The left panel is a diagram showing the isolation procedures of ookinete ghost (SPMTs cytoskeleton) and solute. 5.0 × 106 ookinetes were treated with the ionic detergent sodium deoxycholate (SDC), and DHC3 (HA) and SPMTs (α- and β-Tubulin) were analyzed via IFA in the dhc3::6HA ookinetes and ookinete ghosts. Three independent experiments. Scale bars: 5 μm. H SPMTs co-immunoprecipitated with HA-tagged DHC3 in the dhc3::6HA ookinetes. Co-immunoprecipitation was conducted using the anti-HA antibody. BiP as the loading control. Two independent experiments. I SPMTs co-immunoprecipitated with Myc-tagged DHC3 in the 4Myc::dhc3 ookinetes. Co-immunoprecipitation was conducted using the anti-Myc antibody. BiP as the loading control. Two independent experiments. Source data are provided as a Source Data file.
