Figure 4
Functionally essential residues lie within the Pf CENP-C dimerization domain. (A and B, i-iii) Complementation and ten-fold serial dilution assays with the Pf CENP-C dimerization domain (DD) carrying mutations at the conserved residues essential for the CENP-C dimerization. (i) At 23°C, the different Pf CENP-C-DD mutants, Pf CENP-CF1993A, Pf CENP-CF1996A, Pf CENP-CY2069A, Pf CENP-CN2060A and vector control show normal growth. (ii and iii) At 37°C, both mif2-3 as well as mif2-2 TS mutants expressing the Pf CENP-C-DD mutants, Pf CENP-CF1993A, Pf CENP-CF1996A and Pf CENP-CY2069A show complete growth arrest while the WT Pf CENP-C-DD and Pf CENP-CN2060A show growth rescue. The red boxes show the comparative growth patterns of WT Pf CENP-C-DD and various Pf CENP-C-DD mutants in mif2-3 and mif2-2 TS mutants at 37°C. (C) Confocal images of various Pf CENP-C-DD mutants, Pf CENP-CF1993A, Pf CENP-CF1996A and Pf CENP-CY2069A show severe phenotypes with deformed bud morphology and dispersed and /or fragmented nuclear staining in the TS mutants while the Pf CENP-CN2060A show normal phenotype at 37°C. (D and E) The functional complementation assays with Pf CENP-CP2051L corresponding to mif2-3 TS MIF2pP505L mutation shows growth arrest at 37°C while the non-mutated WT Pf CENP-C-DD facilitates growth at 37°C in mif2-3 TS mutant cells (highlighted with red box). (F) The confocal images representing the gain-or loss-of-phenotypes of mif2-3 TS mutant cells expressing Pf CENP-CP2051L. At 23°C, Pf CENP-CP2051L expressing TS mutants show both proper chromosome segregation (red arrows) as well as chromosome mis-segregation phenotype (black arrow) evident by the nucleus (blue) localization. At 37°C, Pf CENP-CP2051L expressing TS mutants show abnormal phenotypes and most of the mif2-3 cells expressing Pf CENP-CP2051L are unviable. The WT non-mutated Pf CENP-C-DD displays normal chromosome segregation phenotypes at both 23 and 37°C, respectively.