Investigating intraflagellar transport in Leishmania mexicana using dual-colour high-frame rate imaging and genetically encoded fluorescent proteins

Thesis

The assembly, maintenance and function of motile flagella is achieved by two sets of motor protein-driven movement. Inner and outer dynein arm slide adjacent microtubules of the axoneme to produce flagellar bending, whilst the bidirectional movement of intraflagellar transport (IFT) trains along the axoneme are driven by kinesins and dyneins. Previously IFT train speeds have been quantified in mechanically immobilised or genetically paralysed flagella, limiting exploration into the relationship between IFT and flagellar motility. L. mexicana is a uniflagellate protozoan parasite that is highly genetically tractable. It undergoes symmetric-tip-to-base beat for forward swimming and an asymmetric base-to-tip beat for reorientation. In this project, we used high-frame-rate dual-colour microscopy and genetically encoded fluorescent tags to visualise IFT in the beating flagella of L. mexicana. This revealed IFT train speed is largely independent of beat type but is influenced by genetic perturbations to flagellar motility. With increased anterograde IFT train speed in slower-swimming mutants and decreased retrograde train speed in mutants with higher flagellar beating frequencies. We noted that train stalling can occur without substrate interaction, however does so less frequently than in flagella immobilised mechanically. We identified novel molecular components of the IFT system, including microtubule-associated proteins putatively acting as IFT train tracks and mitogen-activated protein kinase negative regulators of IFT train speed. In parallel, we explored the effect of light intensity, light wavelength and redox state on flagellar beating and cellular motility, with initial indications that tryparedoxin proteins with flagellar localisation could play a role in flagellar recovery from oxidative stress. Overall, this project highlights the dynamic interplay of IFT, flagellar motility and environmental regulation in the motile flagella of L. mexicana. It provides a foundation for future studies using advanced imaging and genetic tools to further unravel the regulatory networks governing IFT and flagellar function and their broader implications for cellular motility in other systems.

Authors: Gray, S

• DOI: 10.5287/ora-z5e10m6vn
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Leishmania; CRISPR/Cas9; intraflagellar transport; trypanosomes; flagella; microscopy
• Subjects: Genetic engineering; Microscopy