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. Author manuscript; available in PMC: 2019 Sep 25.
Published in final edited form as: Cell Rep. 2019 May 14;27(7):2132–2146.e7. doi: 10.1016/j.celrep.2019.04.038

Figure 6. The V-ATPase and Rhoptry Maturation.

Figure 6

(A) Western blots of total lysates of iΔvha1-HA tachyzoites grown + and − ATc (3 days) and probed for rhoptry bulb proteins ROP4, ROP7, or TgCA_RP. Immature forms are indicated with arrowheads, and mature forms with arrows.

(B) Comparison between the percentage of immature ROP4, ROP7, or TgCA_RP between lysates of iΔvha1-HA parasites grown + or − ATc.

(C) IFAs of iΔvha1-HA tachyzoites grown + or − ATc and probed with αIMC (highlights nascent daughter cells in dividing parasites), αproROP4 (labels immature rhoptries), and αCA_RP (rhoptries). These are super-resolution images showing the labeling with αproROP4 of dividing parasites (αIMC1 labels nascent daughters; left panels) and non-dividing parasites (right panels). Scale bars: 2 mm.

(D) Quantification of PVs with parasites expressing immature and mature rhoptries in iΔvha1-HA tachyzoite vacuoles grown + and − ATc. There was a significant increase of αproROP4 labeling in non-dividing parasites with ATc (red columns).

(E) Routine EM of iΔvha1-HA tachyzoites grown −ATc (left, −ATc) showing normal rhoptries and their characteristic rhoptry neck (arrowheads) and bulb regions (arrows). In iΔvha1-HA +ATc (3 days), mature rhoptries are absent, and accumulation of vesicular immature rhoptry structures was seen (arrows).

(F) Quantification of the EM images showing significant reduction in the number of tachyzoites containing mature rhoptries after treatment with ATc. Approximately 52–73 parasites were enumerated from three trials.

(G) Super-resolution IFA showing that Vha1 encircles nascent rhoptries labeled by proROP4 (1:500) and TgCA_RP (1:1,000) in intracellular parasites. Statistical analysis was done using a Student’s t test, where *p < 0.05, **p < 0.01, and ****p < 0.0001; n.s., not significant.