Kinesin-8B controls basal body function and flagellum formation and is key to malaria transmission.

Mohammad Zeeshan,Declan Brady,Karine G Le Roch,Carolyn A Moores,Sue Vaughan,Rita Tewari,David Jp Ferguson,Edward Rea,Michael Delves,Steven Abel,Emilie Daniel,Alana Burrrell,Anthony A Holder

doi:10.26508/lsa.201900488

Abstract

Eukaryotic flagella are conserved microtubule-based organelles that drive cell motility. Plasmodium, the causative agent of malaria, has a single flagellate stage: the male gamete in the mosquito. Three rounds of endomitotic division in male gametocyte together with an unusual mode of flagellum assembly rapidly produce eight motile gametes. These processes are tightly coordinated, but their regulation is poorly understood. To understand this important developmental stage, we studied the function and location of the microtubule-based motor kinesin-8B, using gene-targeting, electron microscopy, and live cell imaging. Deletion of the kinesin-8B gene showed no effect on mitosis but disrupted 9+2 axoneme assembly and flagellum formation during male gamete development and also completely ablated parasite transmission. Live cell imaging showed that kinesin-8B-GFP did not co-localise with kinetochores in the nucleus but instead revealed a dynamic, cytoplasmic localisation with the basal bodies and the assembling axoneme during flagellum formation. We, thus, uncovered an unexpected role for kinesin-8B in parasite flagellum formation that is vital for the parasite life cycle.

Highlights

Eukaryotic flagella, known as motile cilia, are conserved microtubule (MT)-based organelles that protrude from cells and drive motility of single cells, or the movement of fluid across ciliated tissue (Mirvis et al, 2018)
Based on its known expression in male gametes and a potential function during male gamete development, we first assessed the function of kinesin-8B throughout the Plasmodium life cycle by using a double crossover homologous recombination strategy to delete its gene in the context of a parasite line constitutively expressing GFP (Janse et al, 2006) (Fig S1A)
We examined male and female gametocytes after activation in the exflagellation/ ookinete medium for the development and emergence of male and female gametes

Summary

Introduction

Eukaryotic flagella, known as motile cilia, are conserved microtubule (MT)-based organelles that protrude from cells and drive motility of single cells, or the movement of fluid across ciliated tissue (Mirvis et al, 2018). The differences in the mechanism of axoneme formation are (1) the Plasmodium basal body, from which the flagellum is assembled, does not exhibit the classical nine triplet MT (9+0) (Mirvis et al, 2018), but instead consists of an electron dense amorphous structure within which nine peripherally arranged MTs can sometimes be resolved (Forancia et al, 2015); (2) basal bodies/ centrioles are not present during asexual stages of the Plasmodium life cycle, unlike Toxoplasma and Eimeria where they play a role during asexual division (Ferguson & Dubremetz, 2014) but assemble de novo in male gametocytes (Sinden et al, 1976); (3) the axoneme itself is assembled from the basal bodies within the male gametocyte cytoplasm, and only after its assembly does the axoneme protrude from the membrane to form the flagellum itself; as a result and very unusually, axoneme construction occurs independently of intraflagellar transport (IFT) (Sinden et al, 1976; Briggs et al, 2004). We show that kinesin-8B–GFP is dynamically associated with the basal bodies and with axonemes during their formation and assembly, consistent with an unexpected role in flagellum genesis

Results

Discussion

Ethics statement