Fission of <i>L. major</i> PVs.

<p>(A) Multidimensional image of dividing <i>L. major</i>-DsRed2 amastigotes (red) hosted by macrophages loaded with Lysotracker (green); a lysotracker-positive interface between the parasites is clearly observed at 1:30h. Image acquisition started after 48 h of infection, and the time of acquisition is shown as hh:mm. Blend filter, bar = 2 µm. Isosurfaces a′ and a″ were attributed to replicating amastigotes using the DsRed2 channel. (B) The Lysotracker RFIs surrounding parasites was measured during 10 h of image acquisition. An increase in the Lysotracker signal was detected for one of the dividing amastigotes, whereas the signal remained low in the other amastigote. After 8 h of acquisition, although both amastigotes remained next to each other, they were surrounded by Lysotracker at low intensities, and no Lysotracker-positive vacuolar interface was observed between them. Gray dots in the graphs indicate the time points to which images presented in E are associated. The data are representative of 5 cases with similar results. (C) Multidimensional image of RAW 264.7 macrophages expressing LAMP1-GFP and Rab7-GFP (green) infected with <i>L. major</i>-DsRed2 (red). Arrowheads indicate the complete division of double-occupancy PV into two individual PVs. This process was followed in 5 other multidimensional images and the time period in which amastigotes share a single vacuole before fission was about 100 minutes. Acquisition started after 4 h of intracellular infection and the time of acquisition is shown as hh:mm. MIP filter, bar = 5 µm. (D) The Lysotracker-positive cluster is observed between two dividing parasites (first row, MIP filter). The attribution of an isosurface to the Lysotracker-positive interface (second row, Blend filter) allowed the measurement of its volume at the image time points. The isosurface contains statistic-based color information ranging from cyan (smaller volumes) to magenta (larger volumes). Image acquisition started after 2 h of infection, and the time of acquisition is shown as hh:mm. Bar = 5 µm. (E) The graph presents the volume measured from the cluster between dividing parasites in the multidimensional imaging. Using a z-stack interval of 1 µm, a detection limit for volumetric measurement of this structure was determined to be 5 µm³. Gray dots in the graphs indicate the time points to which images presented in D are associated. These results were reproduced in 5 other cases.