Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal GFP-Strep tagged version of Cap380 and a C-terminal mCherry-tagged version of IMC1c (inner membrane complex protein 1c, putative).
This mutant was obtained by crossing the following (published) mutant lines, RMgm-5519 that expresses a C-terminal GFP-Strep tagged version of Cap380 and RMgm-1122 that expresses a C-terminal mCherry-tagged version of IMC1c (inner membrane complex protein 1c, putative).
Protein (function)
Cap380 (Plasmodium oocyst capsule protein 380) localises to the P. berghei oocyst capsule, and its depletion leads to oocysts that are gradually eliminated
Phenotype
To investigate sporozoite egress in P. berghei by fluorescence microscopy, we used our Cap380/GFP-Strep parasite line (RMgm-5519) to visualise the oocyst capsule. To also visualise sporozoites, we used our parasite line IMC1c/mCherry that stably expresses the sporozoite-expressed alveolin IMC1c fused to mCherry and displays red fluorescence in mature sporozoites (RMgm-1122). To combine these two fluorescent markers in the same oocyst, the two lines were genetically crossed
Additional information
From the paper: 'We found oocysts with red fluorescence on the outside of the capsule near a small opening. It is difficult to determine conclusively if oocyst undergoing excystation have one, or multiple, holes in their capsule, as their detection relies on their positioning relative to the focal plane of the microscope objective, and additional holes may be forming at different times. Nonetheless, in the egress events we observed, both using Vaseline and conventional cover slips, we only ever observed a single opening (n = 20), indicating this is the most common scenario. Egress of merozoites from the red blood cell has also been reported to take place via a single opening in the host plasma membrane and underlying cytoskeleton. It is noticeable that the oocyst shown in the Figure still possesses a substantial sporoblast body, indicating that the oocyst is not yet fully sporulated (i.e. has used up all sporoblast for sporozoite formation) and thus implying that sporulation can continue whilst excystation is in progress.'
From the paper: 'To study the oocyst capsule during sporozoite egress, we examined Cap380/GFP-Strep oocyst-infected midguts at two weeks post-infected blood feed, when oocyst sporulation is considered to be at its peak. In addition to intact oocysts, live fluorescence microscopy revealed the presence of flattened capsules presumably resulting from oocysts vacated, or being vacated, by their sporozoites. Notably, these empty capsule structures were not visible when midguts were examined by phase contrast (i.e. without capturing GFP fluorescence). This demonstrates that the oocyst does not need to be intact or alive for the capsule to fluoresce, consistent with the capsule constituting a three-dimensional, supramolecular, extracellular matrix that retains water and homeostatic balance.'
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