Lemos, M. | bioRxiv Science

bioRxiv2018

Bidirectional intraflagellar transport is restricted to only two microtubule doublets in the trypanosome flagellum

Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. Here we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3-4 and 7-8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occur on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3-4 seconds while remaining on the same side of the axoneme.

cell biology↗

bioRxiv2018

Biallelic Mutations in LRRC56 encoding a protein associated with intraflagellar transport, cause mucociliary clearance and laterality defects

Defective motile cilia are responsible for a group of heterogeneous genetic conditions characterised by dysfunction of the apparatus responsible for generating fluid flows. Primary ciliary dyskinesia (PCD) is the prototype for such disorders and presents with impaired pulmonary mucus clearance, susceptibility to chronic recurrent respiratory infections, male infertility and laterality defects in about 50 % of patients. Here we report biallelic variants in LRRC56 (also known as ODA8), identified in two unrelated consanguineous families. The phenotype comprises laterality defects and chronic pulmonary infections. High speed video microscopy of cultured patient epithelial cells showed severely dyskinetic cilia, but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link to IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localises with IFT trains and facilitates the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56 null mutations, or a mutation (p.Leu259Pro) corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease, and suggest this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.

genetics↗