Additional remarks phenotype | Mutant/mutation
In the 'promoter swap mutant': the promoter of kinesin13 has been replaced with the asexual blood stage promoter clag9 ((PBANKA_0836300). This promoter is active in asexual blood stages but not in gametocytes.
Protein (function)
Kinesins are microtubule (MT)-based motor proteins that use energy from the hydrolysis of ATP and function in various cellular processes including intracellular transport, mitotic spindle formation and chromosome segregation during cell division, and the organisation of cell polarity and cytoskeleton associated with motility.
There are 14-16 kinesin subfamilies identified in eukaryotes, and categorised based on analysis of the motor domain, with similar biological roles established by in vitro studies, and in vivo phenotypes in these organisms . Kinesin subfamilies that regulate MT dynamics, such as kinesin-8 and -13, are found in most eukaryotes including primitive and evolutionarily divergent eukaryotes.
In a bioinformatic analysis of kinesins in Apicomplexa, nine kinesins encoded in the Plasmodium berghei genome were identified, with members of three conserved kinesin subfamilies (kinesin-5, -8B, -8X and -13); kinesin-4, -15 and -20; and two Apicomplexa-specific kinesins: kinesin-X3 and -X4 (Zeeshan et al., 2019).
Phenotype
To investigate the function of Kinesin-13 in mosquito life cycle stages, we used a promoter trap double homologous recombination (PTD) approach to downregulate gene expression at the blood stage by placing the kinesin-13 gene under the control of the Clag9 promoter. Clag9 is known to be highly expressed in asexual blood stages, but not during sexual differentiation and in mosquito stages.
Knock-out of the kinesin-13 gene is not possible since it has an essential role in asexual blood stages (see RMgm-5010). In the promoter swap mutant Kinesin-13 is expressed in asexual blood stages but is down-regulated in gametocytes and mosquito life cycle stages.
Normal asexual blood stage growth/multiplication and production of gametocytes.
Strongly reduced male gamete formation (exflagellation). Strongly reduced zygote/ookinete formation. No oocyst or sporozoite formation.
Evidence is presented that: 'High resolution ultrastructure analysis of kinesin-13PTD parasites indicates defects in the spindle assembly and axoneme microtubules (MT) of gametocytes, and the subpellicular MT of ookinetes'.
Additional information
From the paper: 'we used an auxin-inducible degron (AID) system to try and study the effect of rapid kinesin-13 degradation in gametocytes. We tagged the endogenous kinesin-13 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin (Philip and Waters, 2015), and successfully generated kinesin-13- AID parasite lines as shown by integration PCR but could not deplete kinesin-13 protein by auxin treatment'.
Other mutants |