Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of kinesin-5 and expresses GFP under the constitutive eef1a promoter
Protein (function)
Kinesin-5 proteins are a family of molecular motors that is structurally and functionally conserved throughout eukaryotes. They are involved in spindle pole separation and are considered essential for mitosis in the vast majority of eukaryotes. Kinesin-5 is located at spindle MTs and spindle poles during cell division and is distributed diffusely in the cytoplasm during interphase in most eukaryotic cells.
In Plasmodium berghei there are nine kinesin genes, including two kinesin-8 genes that are important in cell division and male gamete formation. There is a single Plasmodium kinesin-5.
Phenotype
Normal blood stage development, gametocyte and ookinete production. There was no significant difference in the number of Δkinesin-5 and wild type WT-GFP oocysts, and the size of the oocysts was similar for both parasites. However, we observed a 40 to 50% decrease in the number of sporozoites in each oocyst at days 14 and 21 post-infection in Δkinesin-5 parasites compared to WT-GFP parasites. A significant decrease in the number of Δkinesin-5 sporozoites in salivary glands was also observed, but the shape, size, and motility of these sporozoites were indistinguishable from WT-GFP parasites. Normal infectivity of sporozoites as determined by blood stage infection in mice after infection by mosquito bite
Additional information
From the Abstract: 'Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella.'
To quantify the expression of kinesin-5 at different stages of the parasite life cycle, we isolated RNA and performed qRT-PCR. Kinesin-5 is expressed constitutively throughout the blood and mosquito stages of parasite development, with the highest level in gametocytes, followed by schizonts and ookinetes
Analyses of a mutant expressing a C-terminal GFP-tagged version of kinesin-5 (RMgm-4875) provided evidence that:
- Pbkinesin-5 is located at the spindle apparatus during mitotic stages of asexual blood stage schizogony
- Spatiotemporal dynamics of Pbkinesin-5 reveal its location on the spindle apparatus during male gametogony
- During meiosis in zygote to ookinete development, Pbkinesin-5 location follows spindle dynamics. Kinesin-5-GFP fluorescence was initially diffuse within the zygote nucleus, after 1.5 to 2 hours post fertilization the GFP signal coalesced to a single focal point adjacent to the DNA
- Pbkinesin-5-GFP exhibits multiple nuclear foci during oocyst development and in liver stage schizogony
Other mutants |