Figure 7. PP1 ensures rapid Ca²⁺ mobilization prior to zaprinast-induced egress.

(A) Selected frames of time-lapse images of PP1-AID parasites expressing the genetically encoded Ca²⁺ indicator GCaMP following treatment with 500 µM zaprinast. (B) Normalized GCaMP fluorescence of individual vacuoles was tracked after zaprinast treatment and prior to egress (opaque lines). The solid line represents the mean normalized fluorescence of all vacuoles across n=3 biological replicates. (C) The time to maximum normalized fluorescence of individual vacuoles after zaprinast treatment. Different replicates are shown in different shades of gray. Small points correspond to individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (D) Selected frames of time-lapse images of PP1-AID parasites expressing the genetically encoded Ca²⁺ indicator GCaMP following treatment with 4 µM A23187. (E) Normalized GCaMP fluorescence of individual vacuoles was tracked after A23187 treatment and prior to egress (opaque lines). The solid line represents the mean normalized fluorescence of all vacuoles across n=3 biological replicates. (F) The time to maximum normalized fluorescence of individual vacuoles after A23187 treatment. Small points correspond to individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (G) Fluorescence intensity of Fura2/AM-loaded TIR1 or PP1-AID parasites treated with IAA for 5 hr before and after addition of the 1.8 mM Ca²⁺. Representative traces from three biological replicates. (H) Resting cytoplasmic [Ca²⁺] prior to incubation in buffers with elevated [Ca²⁺]. p Values were calculated from an ANOVA. (I) The rate of Ca²⁺ entry in the first 20 s after addition of 1.8 mM Ca²⁺ to parasites p values were calculated from an ANOVA. Entry rates following addition of other concentrations are shown in Figure 7—figure supplement 1.

Figure 7—figure supplement 1. PP1-AID parasites egress, as quantified by video microscopy.

(A) The time to vacuole egress after zaprinast treatment was manually scored. Different replicates are shown in different shades of gray. Small points represent individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (B) The normalized fluorescence change of individual vacuoles after zaprinast treatment. Different replicates are shown in different shades of gray. Small points correspond to individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (C) The time to vacuole egress after A23187 treatment was manually scored. Different replicates are shown in different shades of gray. Small points correspond to individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (D) The normalized fluorescence change of individual vacuoles after A23187 treatment. Different replicates are shown in different shades of gray. Small points represent individual vacuoles; large points are the mean for each replicate. p Values were calculated from a two-tailed t-test. (E) Ca²⁺ entry rates corresponding to (F). The slope of the trace at the time of addition of Ca²⁺ was measured as the change in the concentration of Ca²⁺ during the initial 20 s after addition of Ca²⁺. Each bar represents the average of a minimum of three biological replicates. ANOVA was used for the statistical analyses. * p<0.01. (F) Fluorescence intensity of Fura2/AM-loaded TIR1 or PP1-AID parasites treated with IAA for 5 hours upon incubation with buffers of the indicated [Ca2+]. Representative traces from at least three biological replicates.