Additional remarks phenotype | Mutant/mutation
Parasites expressing GFP-tagged SAS4 (RMgm-5132) were crossed with parasites expressing mCherry-tagged kinesin-8B (RMgm-4844). After crossing gametocytes were analysed that express both SAS4-GFP and kinesin-8B-mCherry (SAS4-GFP/kinesin-8B-mCherry).
Protein (function)
SAS4 is a marker of the basal body (BB). SAS4/SAS6 is an ancestral core proteins involved in BB biogenesis.
kinesin-8B: 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
From the paper:
A hallmark stage post-activation of gametocytes is the assembly of axonemes which are internally coiled around the nucleus and can clearly be seen at ~3-4 mins after activation using live cell imaging microscopy and basal body marker SAS-GFP with an axoneme marker Kinesin8B-mcherry.
Additional information
We analysed the positioning of the 8 NPs within the nuclear envelope. As the cell undergoes three rapid rounds of DNA replication, spindle microtubules nucleate from each NP and chromosome segregate as observed with kinetochore marker NDC80. The basal body marker SAS4-GFP is found associated with the kinetochore marker NDC80-Cherry even at 8 minutes after activation when nuclear content is octoploid as observed by live cell imaging. (see RMgm-5364).
From the Abstract:
Upon activation, the male P. berghei gametocyte undergoes three rounds of inter- nuclear mitosis and assembles eight basal bodies and axonemes around the nucleus prior to ex-flagellation, resulting in 8 flagellated male gametes in 12-15 minutes. In this study we used serial block face scanning electron microscopy (SBF-SEM) and cellular electron tomography (ssET) of P. berghei microgametocytes to examine the 3D architecture of key structures during male gamete formation. Our data has revealed an exquisite organisation of axonemes coiling around the nucleus in opposite directions forming a central ‘axoneme band’ in microgametocytes. Furthermore, we discovered that the nucleus of microgametes is tightly coiled around the axoneme in an exquisitely complex structure whose formation starts before microgamete emergence during ex-flagellation.
Other mutants |