Fig 2.

The N-terminal extension of CDPK2A influences its localization and is necessary for parasite egress. (A) Schematic representation of complementing constructs with comparison to CDPK2A genomic locus. Dotted lines indicate gaps in the sequence where introns have been removed. Shaded region encoding the kinase domain. 3-MB-PP1-sensitive alleles are highlighted in red. (B) Immunoblot of complemented strains probing for the endogenous allele with Ty and the complementing allele with HA. (C) Immunofluorescence microscopy of endogenously Ty-tagged CDPK2A^M and CDPK2A^G and HA-tagged complementing copies of CDPK2A^M. Merged image displays Ty (green), HA (magenta), DNA (blue), and phase (gray scale). Scale bar is 10 µm. (D) Kinetic traces of A23187-stimulated parasite egress. Graphs are egress of 3-MB-PP1-treated parasites as percentage of the vehicle. A23187 was added 1 second after the start of imaging. Line plots represent mean ± SEM for n = 3 biological replicates. (E) Maximum egress achieved by each strain during the observation window, displayed as percentage of the vehicle. Bars represent mean ± SEM of n = 3 biological replicates; significance was calculated by unpaired one-tailed t-test. (F) Kinetic traces of zaprinast-stimulated parasite egress. Graphs are egress of 3-MB-PP1-treated parasites as percentage of the vehicle. Zaprinast was added 1 second after the start of imaging. Line plots represent mean ± SEM for n = 3 biological replicates. (G) Maximum egress was achieved by each strain during the observation window, displayed as percentage of the vehicle. Bars represent the mean ± SEM of n = 3 biological replicates; significance was calculated by unpaired one-tailed t-test.