Fig. 7
Transient self-association and mobility of actin bundles. a Representative SIM movie shows transient association and circular flow of actin bundles in the RB of a PV. Detailed view showing SIM frames of the RB. Bundles associate transiently with each other following circular flow in the residual body. The experiment was performed using transgenic parasites expressing actin-Cb-em (orange) and Myo-SNAP visualised with ligand TMR (cyan). b Kymograph analyses from the RB (in A) demonstrates that F-actin flow is stationary (blue), anterograde (red) and retrograde (green). Quantitative display of actin flow from measurements in multiple regions of a RB. Visualisation of converging actin flow from multiple parasites supports transient association of F-actin filaments and the formation of the circular scaffold in the RB. c 3D-SIM images of the F-actin network. The depth information is colour coded. Data show variable and complex actin bundle association. Representative images are taken from the actin network connecting parasites from three PVs. The experiment was performed on transgenic parasites expressing actin Cb-em. d SIM image of an F-actin bundle (yellow) decorated with MIC2-HALO (magenta). A strain stably expressing MIC2-HALO was transfected with actin Cb-SNAP. Ligand for HALO and SNAP were Oregon green and TMR respectively. e Representative SMLM image of F-actin bundles connecting parasites in the PV. A PALM experiment was performed using a cell line stably expressing actin-Cb tagged with photoconvertible monomeric mEos3.2 protein (Cb-mEos3.2). This method was a complementary imaging approach to SIM, and was used to resolve the composition of the actin network connecting parasites inside the PV. Variable diameters measured along the actin filamentous structure indicated that the network is an actin bundle consisting of variable number of short F-actin filaments. f Representative SMLM image of F-actin bundles forming the RB. A PALM experiment was performed using a cell line stably expressing Cb-mEos3.2. The results show variable diameters of F-actin bundles demonstrating that the RB consists of a network made of bundles with variable number of F-actin filaments. Scale bar is 5 µm. Time in movie frames expressed in minutes. Source data are provided as a Source Data file
