Apicomplexan F-actin is required for efficient nuclear entry during host cell invasion (2019)
Figure 1

Figure 1. F-actin, formed at two major nucleation centres forms a dynamic, continuous network within the cytosol of the parasite.

A. Stills depicting actin flow (see movie EV1) in extracellular RH, myoA KO and adf KD parasites expressing Cb EmeraldFP. Continuous F-actin flow can be seen from the apical tip, to the Golgi region towards the posterior pole of the parasite. No difference can be observed between RH and myoA KO. For adf KD, the flow is completely abrogated. Red arrowhead marks the Golgi region, while the yellow arrowhead marks the apical nucleation centre. The time-lapse is presented in Movie EV1.

B. Skeletonisation processing of (A) depicting areas where F-actin dynamics/flow is prevalent. Individual signals for F-actin form a continuous, dynamic network that connects apical and posterior pole of the parasite. Red arrowhead marks the Golgi region nucleation centre, while the yellow arrowhead marks the apical nucleation centre. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin. The time-lapse is presented in Movie EV1.

C. Kymograph analysis of (A) and (B). The kymograph was generated by tracing a line in the middle of the parasite's body (red line). The colour-coded kymograph represents forward movement (red), backwards movement (green) and static F-actin (blue). The results demonstrate continuous exchange of F-actin from the apical to the posterior pole of the parasite within the cytosol of the parasite. This cytosolic exchange is similar in RH and myoA KO parasites, while completely abolished in the case of adf KD.

D. Top three rows: Parasites expressing Cb EmeraldFP along with myoA-SNAP before and after Ca2+-Ionophore (A23187) or BIPPO treatment. The parasites were also treated with 2 µM of Cytochalasin D or DMSO (as control) for 30 min. After addition of A23187, preferential relocalisation of actin can be observed at the apical tip, while addition of BIPPO caused F-actin accumulation at the basal end. 4th row: myoAKO parasites expressing Cb-EmeraldFP before and after treatment with A23187 and BIPPO. While no apical or basal accumulation of F-actin is observed, some peripheral location of F-actin occurs in presence of A23187 or BIPPO, preferentially in the apical half of the parasite. Bottom row: adf cKD parasites expressing Cb-EmeraldFP before and after treatment with Ca2+ Ionophore or BIPPO. No apparent change in F-actin localisation can be seen upon treatment. The time-lapse is presented in Movie EV2.

E. Skeletonization of movies shown in D. Before addition of A23187 or BIPPO, RH and myoA KO parasites behave similar with no accumulation of F-actin at the apical tip. After treatment with calcium ionophore A23187, preferential accumulation of F-actin at the apical tip can be observed for RH (red arrowhead), while BIPPO presented preferential accumulation in the basal end (red arrowhead). In myoA KO, some relocalisation of actin is observed at the periphery of the parasite (yellow arrowhead). In the case of adf cKD, no relocalisation occurs. The images represent a projection of frames for each timepoint to provide a better overview of the dynamics of F-actin.

F. Quantification of actin accumulation before and after treatment with Ca2+ Ionophore or BIPPO. The parasites were counted for F-actin accumulation after adding Ca2+ Ionophore or BIPPO. Numbers were generated by counting total number of parasites and then number of parasites with actin accumulation on the apical or basal tip. A minimum of 200 parasites were counted upon 3 biological replicates. Two-way ANOVA was used for statistical analysis and Tukey's multiple comparisons test. **** p value <0.0001. Error bars represent standard deviation.

Data information: Numbers indicate minutes:seconds; and Scale bars represent 5 µm.