LAX28 is required for the stable assembly of the inner dynein arm f complex, and the tether and tether head complex in Leishmania flagella

Motile eukaryotic flagella beat through coordinated activity of dynein motor proteins; however, the mechanisms of dynein coordination and regulation are incompletely understood. The inner dynein arm (IDA) f complex (also known as the I1 complex), and the tether and tether head (T/TH) complex are thought to be key regulators of dynein action but, unlike the IDA f complex, T/TH proteins remain poorly characterised. Here, we characterised T/TH-associated proteins in the protist Leishmania mexicana. Proteome analysis of axonemes from null mutants for the CFAP44 T/TH protein showed that they lacked the IDA f protein IC140 and a novel 28-kDa axonemal protein, LAX28. Sequence analysis identified similarities between LAX28 and the uncharacterised human sperm tail protein TEX47, both sharing features with sensory BLUF-domain-containing proteins. Leishmania lacking LAX28, CFAP44 or IC140 retained some motility, albeit with reduced swimming speed and directionality and a propensity for flagellar curling. Expression of tagged proteins in different null mutant backgrounds showed that the axonemal localisation of LAX28 requires CFAP44 and IC140, and the axonemal localisations of CFAP44 and IC140 both depend on LAX28. These data demonstrate a role for LAX28 in motility and show mutual dependencies of IDA f and T/TH-associated proteins for axonemal assembly in Leishmania

CRISPR/Cas9 gene editing in the West Nile Virus vector, Culex quinquefasciatus Say.

Culex quinquefasciatus Say is an opportunistic blood feeder with a wide geographic distribution which is also a major vector for a range of diseases of both animals and humans. CRISPR/Cas technologies have been applied to a wide variety of organisms for both applied and basic research purposes. CRISPR/Cas methods open new possibilities for genetic research in non-model organisms of public health importance. In this work we have adapted microinjection techniques commonly used in other mosquito species to Culex quinquefasciatus, and have shown these to be effective at generating homozygous knock-out mutations of a target gene in one generation. This is the first description of the kmo gene and mutant phenotype in this species