TgMAPK-L1 has a key role in coupling mitosis and cytokinesis.
(A) In replicating parasites endogenously tagged wild-type TgMAPK-L1HA (red stain) showed a predominant alignment with single (upper panel) and duplicated (lower panel) centrocone (Cc; anti-MORN1 staining), surrounded by the TgMORN1-rings (new daughter’s basal complexes). Non-color guide panels of the inverted merged images of the anti-IMC1 (blue channel) and the anti-MORN1 (green channel) staining were included here to provide a reference to the location and scale of the subcellular structures observed within the parasite. Magnified merged images to the right of each panel are included to provide greater detail of the key structures stained. (B) Amplified inner cores (TgCEP250-L1HA) in ts-TgMAPK-L1 mutant 11–31G12 parasites arrested at 40°C for 20 h maintained alignment with the nuclear centrocone (anti-TgMORN1 staining); however, MORN1-rings (daughter’s basal rings) were no longer attached to duplicated centrocones in many parasites. Asterisks indicate position of the basal complex of the mother cell and “<” points to the aligned inner core/centrocone structures. Examples of disconnected basal rings are evident in the lower 40°C panel (long arrow), while at the permissive temperature (34°C) the daughter’s basal rings are tightly connected to the centrocone. Yellow dashed line in the merged images indicates the parasite size (40) or the size of the vacuole (34). (C) Bar graph shows quantification of the disruption of the daughter’s MORN1-ring attachment to the centrocone. Only S/M stages that normally display MORN1-rings were quantified at 34°C. Vacuoles with detectable MORN1-rings were analyzed at 40°C. No less than 100 vacuoles were analyzed for each condition (for all raw data see S1 Data). (D) Co-staining of the nuclear centrocone and basal rings (anti-TgMORN1 antibody, green) with the membrane scaffold (anti-IMC1 antibody) in the ts-TgMAPK-L1 parasites arrested at 40°C for 20 h. Merged images are shown in the upper panel. Anti-TgMORN1 stain in the Guide panel highlights only the centrocone and basal ring relationships. The three images are representative of the predominant morphological defects associated with ts-TgMAPK-L1 in mutant parasites. First image represents an early step in the development of phenotype: mother cell with multiplied centrocones (7) and no visible formation of the daughter’s basal rings (“^”). The mother cell in the second image similarly has amplified centrocones (4), however, they are connected to the well-developed daughter MORN-rings (arrows). The consecutive step of abnormal multiple budding (7) is represented on the third image. Note that each daughter cell has a constricted basal ring (arrow) at the proximal end of the body.