Defining the molecular composition and organisation of Transition fibres in Trypanosoma brucei and their roles in flagellar assembly and length control

The eukaryotic cilium/flagellum is a slender microtubule-based projection that extends, with a contiguous membrane, out from the cell body. They possess a highly conserved core structure, composed of a microtubule-based axoneme that runs through its core. The axonemal microtubules are nucleated by a barrel shaped microtubule-organising basal body. Transition fibres (TFs) are appendage structures surrounding the distal end of the mature basal body that enable membrane docking and regulate the trafficking of intraflagellar transport (IFT) proteins required for flagellar assembly. Transition fibres are present in all ciliated organisms yet only a handful of TF proteins have been identified in mammalian cells. We hypothesise that there are a number of additional components to be identified. We have identified 35 putative TF proteins and used automated image analysis to map their spatial arrangement based on their diameter and offset from the mature basal body. We confirmed the localisation of these proteins at the transition fibre by co-localisation with known transition fibre proteins and investigated transition fibre dynamics and assembly by analysing the recruitment tagged proteins over the cell cycle. We assessed the function of 20 transition fibre proteins by RNAi and found six proteins that are indispensable for flagellar assembly. We also identified 2 proteins that appear to be involved in flagellar length control and a further 2 proteins that appear to act as regulators of length control. We identified two transition fibre proteins that exhibit a phenotype relating to cell cycle progression but not related to flagellum formation. Ciliopathies are a class of diseases related to the dysfunction of cilia. We also investigated the mechanism of four human ciliopathy orthologues (OFD1, CEP90, RABL2B and CEP19) in the import and loading of IFT onto the flagellar axoneme. The study provides evidence that the protein network of the TFs is far more complex and intricate than what has been previously described in mammals. We also show that trypanosome transition fibres are indispensable for both flagellar assembly and length control whilst also potentially playing further roles in cell cycle progression. We have described a novel mechanism of human ciliopathy protein OFD1 in ciliary assembly that has not been discovered previously. We conclude that transition fibres are essential structures in the context of cilium/flagellum assembly and length control that contribute directly to human ciliopathies and we have leveraged the trypanosome model effectively to address broad biological questions that relates closely to human disease.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2022

Author dc:creator

• Ahmed, Manu

Contributors dc:contributor

• Vaughan, Sue

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/ekr1-dj60

OAI identifier oai:identifier

tle:6cf5bc47-89e0-4791-a98a-05388d79bcd2:d6bd9758-527a-46cd-bfe2-c433766e8fca:1