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. 2023 Aug 23;14(5):e01358-23. doi: 10.1128/mbio.01358-23

Fig 5.

Fig 5

CDPK2A impacts plaque formation, gliding motility, and microneme discharge. (A) Depletion of each of the three CDPKs affected plaque formation differently: parasites depleted of CDPK1 formed no plaques, those depleted of CDPK2A formed fewer plaques, and those depleted of CDPK3 plaqued normally. Images are representative of n = 4 biological replicates. Scale bar is 400 µm. (B) CDPK2A-AID parasites form smaller plaques than the TIR1 parental line, and the effect is exacerbated by the depletion of the kinase (+ IAA). Scatter plot displays areas for >270 individual plaques per sample; mean ± SD is overlaid; P-value was calculated by unpaired t-test. (C) CDPK2A depletion does not impact parasites’ ability to invade host cells. Bars represent the mean ± SEM for n = 3 biological replicates; P-value calculated by paired t-test. (D) 3D gliding motility in Matrigel following the depletion of each kinase. Maximum intensity projections of tracked parasites. Scale bar is 100 µm. (E) Proportion of parasites observed moving in 3D gliding motility assays. Each point is the mean of three technical replicates with 634–1,500 observations per condition; bars represent the mean ± SEM of n = 3 biological replicates; significance was calculated by unpaired one-tailed t-test. (F) Proportion of parasites observed moving in 3D gliding motility assays of CDPK2A-AID-HA parasites with and without IAA. Each point is the mean of the technical replicates; bars represent the mean ± SEM of n = 4 biological replicates; significance was calculated by paired t-test. (G) 3D gliding motility in Matrigel following the depletion of CDPK2A-AID-HA. Maximum intensity projections of tracked parasites. Scale bar is 100 µm. (H) Depletion of CDPKs decreases microneme discharge. Gaussia luciferase activity, from a microneme-localized construct, was assayed in supernatants following 30 minutes of stimulation with FBS and zaprinast. Points represent individual biological replicates (n = 8 for CDPK1-AID, n = 4 for CDPK3-AID or CDPK2A-AID), with bars indicating mean ± SEM; significance was calculated by paired one-tailed t-test. (I) Proposed model for the relationship between kinases that positively regulate parasite motility.