Key roles for kinesin-13 and kinesin-20 in malaria parasite proliferation, polarity 1 and transmission revealed by genome-wide functional analysis 2

Kinesins are microtubule-based motors important in cell division, motility, polarity, and 50 intracellular transport in many eukaryotes. However, they are poorly studied in the 51 divergent eukaryotic pathogens-Plasmodium spp. , the causative agents of malaria, 52 which manifest atypical aspects of cell division and plasticity of morphology throughout 53 the lifecycle in both mammalian and mosquito hosts. Here we describe a genome-wide 54 screen of Plasmodium kinesins, revealing diverse subcellular locations and functions in 55 spindle assembly, axoneme formation and cell morphology. Surprisingly, only kinesin-13 56 is essential for growth in the mammalian host while the other eight kinesins are required 57 during the proliferative and invasive stages of parasite transmission through the 58 mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in 59 microtubule dynamics during apical cell polarity formation, spindle assembly, and 60 axoneme biogenesis. These findings help us to understand the importance of 61 microtubule motors and may be exploited to discover new therapeutic interventions 62 against malaria. 63 64 65 66 Here, we present a comprehensive genome-wide screen of all P. kinesins, including additional analyses of previously studied kinesin-5, -8B and -8X (18- 121 20), using gene-targeting approaches, live-cell imaging, ultrastructure expansion microscopy and electron microscopy, and RNA-seq and ChIP-seq analyses. We examine the subcellular location of each kinesin using a protein endogenously tagged at the C-terminus with GFP, revealing a differential localisation of kinesins in mitotic and meiotic stages and a pellicular and polar location in certain invasive stages. Eight of the nine kinesin genes are required only for parasite transmission through the mosquito vector, during the sexual and sporogony stages. Only kinesin-13 is likely essential during blood stage schizogony. An in-depth analysis of kinesin-13 and -20 during gametocyte and ookinete stages revealed distinct subcellular locations and functions in MT spindle assembly and formation, axoneme assembly and cell polarity. Kinesin-20 was 131 associated with a striking ring-like structure during zygote to ookinete differentiation and deletion of the kinesin-20 gene revealed a function in the morphology and motility of the ookinete. Kinesin-13 is expressed at all proliferative stages of the life cycle, and it associates with the kinetochore. A kinesin-13 genetic knockdown affected MT dynamics during spindle formation and axoneme assembly in male gametocytes, and subpellicular MT organization in ookinetes. These findings help us understand the importance of MT motors and may be exploited to discover new therapeutic interventions against malaria. axoneme the kinesin-8B gene defective basal body formation and axoneme assembly during nuclear the gene found a cdc2-related kinase (CRK5) for nuclear spindle formation but has no effect on axoneme assembly during male gametogenesis Previous studies a similar phenotype for CDPK4 and MAP2 gene Giardia MT both flagellar note the (M) ( e) Details longitudinal sections through the showing similar substructures consisting of three conoidal rings (CR), an anterior polar ring 1 (P1) formed by the IMC and a second polar ring (P2) representing the initiation site for the sub-pellicular microtubules (Mt). The polar rings are separated by a collar consisting of an outer electron dense layer (cd) and an inner electron lucent layer (cl). Note the micronemes (M) with ducts (D) direct to the anterior plasmalemma. ( g) Cross section through the periphery of the anterior complex of a WTGFP (f) and a kinesin-20 (g) parasite showing similar sub-structure consisting of the outer plasmalemma (PM) and the underlying inner membrane complex (IMC) which appears fused to the outer electron dense (cd) region of the apical collar while the more electron lucent inner region is in close contact with sub- pellicular microtubules plasmalemma Note parasite