Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of kinesin-8B
Protein (function)
Plasmodium axonemes display a classical “9+2” structure: 9 doublets of microtubules arranged in a circular pattern surrounding a central pair of singlet microtubules. Their mode of assembly is unusual: Plasmodium axonemes are assembled in the cytoplasm of the microgametocyte independent of intraflagellar transport, a bidirectional transport machinery essential for the construction of flagella in most other eukaryotes. The components of the axoneme, such as tubulin, are ubiquitous in the cytoplasm and are assembled upon activation. The nuclear envelope does not break down during the 3 mitotic replications. The axonemes are however linked to the 8 replicated haploid genomes in the nucleus through spindle poles situated in nuclear pores, thus forming 8 flagellated motile microgametes, which, following violent axonemal ‘swimming’ are expelled from the microgametocyte in an exceptionally fast process called “exflagellation”. These dynamic processes will certainly require motor proteins, such as kinesins and dyneins, to transport cargoes, assemble the structure and generate the force for motility. Most dyneins involved in motility, are part of the complex organisation of outer and inner dynein arms present on each doublet microtubule. In Plasmodium, inner dynein arms are seen less often than outer dynein arms and appear thinner in electron microscopy studies.
Only 10 kinesin encoding genes are found in the P. berghei genome, fewer than in other eukaryotes. Among the kinesins identified by proteomic studies of sexual stages, only three are present in male gametocytes and gametes. According to the comprehensive study of kinesins across eukaryotes by Wickstead et al. (2010), they belong to the families kinesin-8 (subfamily 8B), -13 and -15. Those kinesins are among the microtubule motors phosphoregulated during P. berghei gametocyte activation. While most kinesins display predominantly one activity, either transport along microtubules or depolymerization, kinesin-8 family members are reportedly multitalented. They can walk on microtubules, but also regulate microtubule length, during mitosis and/or ciliary and flagellar assembly.
PbKIN8B encompasses a classical motor domain positioned centrally [aa 779- 1118] with 8 predicted ATP binding sites [aa 787, 872, 875, 877, 878, 879, 880, 1018] and 3 predicted microtubule interaction sites [aa 1071, 1074, 1077]. With the exception of the kinesin motor domain and of coiled-coil motives in the C-terminal region, no additional protein signatures or nuclear localisation signals (NLS) were detected.
Phenotype
Analysis of a mutant lacking expression of kinesin-8B (RMgm-4682) showed the following: Normal production of male and female gametocytes. No male gamete formation as shown by absence of exflagellation centers. Strongly reduced fertilisation, ookinete and oocyst formation.
Analysis of Pbkin8B-gfp parasites showed: Normal production of male and female gametocytes. Normal male and female gametocyte production. No GFP expression in blood stages and female gametocytes. In unactivated male gametocytes, cytosolic expression. Upon activation, the localization of the Pbkin8B-gfp signal changes and is seen as punctiform lines with spatial and temporal dynamics similar to the axonemal marker α-tubulin II. Expression in male gametes.
Additional information
Other mutants
a mutant lacking expression of kinesin-8B (RMgm-4682)
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