FIG 6.
The conserved region of coiled-coil 4 is essential for IMC32 function. (A) Diagram of IMC32ΔCC4-5 in IMC32cKD parasites. Without ATc, IMC32ΔCC4-5 colocalizes with the endogenously 3×Ty-tagged IMC32 (WT). Red, rabbit anti-HA; green, mouse anti-Ty. Upon addition of ATc, endogenous IMC32 is lost, and costaining with IMC6 reveals a collapse of the IMC. Red, mouse anti-Ty; green, rabbit anti-IMC6. (B) Diagram of the three sections within coiled-coil 4 that were individually deleted for IMC32 analysis, labeled A to C. (C) Gene model of IMC32ΔC and IFA showing endogenous IMC32 and IMC32ΔC colocalization in daughter buds. Red, rabbit anti-HA; green, mouse anti-Ty. (D) Quantification of plaque assays with IMC32cKD, IMC32cKD + IMC32FL, and IMC32cKD + IMC32ΔC parasites under conditions with and without ATc. The difference in plaque size between IMC32FL and IMC32ΔC with ATc was significant (58% reduction, P < 0.0001). (E) Gene model of IMC32ΔB and IFA showing endogenous IMC32 and IMC32ΔB colocalization in daughter buds. Red, rabbit anti-HA; green, mouse anti-Ty. (F) Quantification of plaque assays with IMC32cKD, IMC32cKD + IMC32FL, and IMC32cKD + IMC32ΔB parasites under conditions with and without ATc. The difference in plaque size between IMC32FL and IMC32ΔB with ATc was not significant (P ≥ 0.05). (G) Alignment showing similarity of IMC32 residues 769 to 797 with its Plasmodium homologue, PF3D7_0717600. Black highlights indicate identity; gray highlights indicate similarity. (H) Gene model of IMC32ΔA and endogenous IMC32 and IMC32ΔA colocalization in daughter buds. Red, rabbit anti-HA; green, mouse anti-Ty. (I) Quantification of plaque assays with IMC32cKD, IMC32cKD + IMC32FL, and IMC32cKD + IMC32ΔA parasites under conditions with and without ATc. IMC32ΔA parasites were unable to form plaques (P < 0.0001). Scale bars for all images, 4μm.