Building a molecular machine: analysis of co-chaperones for assembly of ciliary dynein motor complexes

Almost every cell of your body has a thin, hair-like outgrowth called a cilium. Some types of cilia are capable of bending or beating and are involved in fluid movement. Such 'motile cilia' are found for example on cells lining our airways for mucus movement as well as the fallopian tubes for wafting a new egg towards the uterus. Moreover, sperm cells swim by means of a beating flagellum, which is essentially a long motile cilium. All these cilia bend through the action of banks of 'motor proteins' within them. These motor proteins form huge molecular machines - some of the largest known in nature. Perhaps not surprisingly, assembling these motor protein machines is extremely complex and requires other dedicated proteins that act as 'molecular chaperones' to ensure they are built correctly during the construction of the cilium by the cell. This proposal concerns the identification of the chaperone proteins and analysing how they function in motor assembly.

The importance of motor assembly is illustrated by what happens when it goes wrong: primary ciliary dyskinesia (PCD) is a human inherited disease in which cilia are immotile due to failure of these motor proteins. The result of this is the patient has symptoms related to infertility and difficulties in clearing mucus, leading for instance to frequent and damaging chest infections. Severe cases also have situs inversus - in which internal organ positioning is disrupted (e.g. the heart is no longer on the left side of the chest). Mutations in many different genes cause PCD. Some mutations are in genes that code for the molecular chaperone proteins. How these chaperone proteins work is not clear. Moreover, the chaperone proteins are not unique to humans - the entire pathway of motor assembly is very ancient, and is found in organisms from protozoa (e.g. the swimming Paramecium) upwards. To further our knowledge of how motor proteins assemble, our strategy is to look in the fruit fly, Drosophila melanogaster. The fruit fly is easy to rear and to study. Sophisticated genetic and cellular approaches can be used in Drosophila to discover genes that are required for motor assembly. We shall examine the effect of disrupting the function of these genes. This is quite straightforward to achieve because in Drosophila, motile cilia are required only for senses and sperm, and so flies with defective motile cilia are easy to spot through obvious sensory deficits and male infertility. Moreover, recent advances in genetic and microscopy tools mean that we can develop much more sophisticated ways of probing the motor assembly pathway, within the context of a whole organism, than has been hitherto possible in animals.

Ease of gene discovery and analysis is not sufficient. Just as important is the fact that the molecular machinery of the cilium is completely conserved between insects and 'higher' animals. Therefore, the chaperone mechanisms discovered in Drosophila are likely to be important in animals and humans too. For studies into cilium biology it is cost-effective and ethically more acceptable to use Drosophila than more complex organisms where possible.

The dynein motor complexes of motile cilia are among the largest molecular machines in the cell. In recent years about 11 'assembly factors' have been identified for the cytoplasmic pre-assembly of dynein motor complexes prior to their trafficking to the cilium. These are hypothesised to be part of a highly conserved chaperone-mediated assembly pathway. To understand this assembly pathway there is a need to (1) discover further assembly factors to complete the 'tool kit' of dynein pre-assembly, and (2) carry out detailed analysis of their function, preferably in a organismal context capitalising on latest advances for genetic and microscopy analysis. Our recent research has firmly established Drosophila as a powerful model for these two aims. In Drosophila, motile cilia are confined to sensory neurons and sperm, which are readily accessible for analysis, and yet the entire known dynein assembly pathway is conserved. Our recent functional genomic screen has led to the identification of new dynein assembly factors (Zmynd10, Heatr2 and Wdr92), and several further (conserved) candidates for which we have excellent preliminary data. We shall systematically analyse CRISPR-generated mutants of all known and new assembly factors to categorise their dynein assembly defects. This will entail our newly developed method of unbiased proteomic detection of protein abundance changes in mutant spermatocytes. Interactions with other proteins (co-chaperones and dynein clients) will be determined by immunoprecipitation of tagged proteins from testes and identification of interactors by mass spectrometry. New tools for the analysis of dynein subunit dynamics (localisation and turnover) will be generated using CRISPR technology to tag endogenous genes, thereby allowing sophisticated microscopy analysis in an in vivo context. This will provide a benchmark for other organisms.

Medical impact

Although this is a basic research proposal, our past identification of new dynein assembly factors in Drosophila has successfully facilitated discovery of new mutations causing Primary Ciliary Dyskinesia (ZMYND10 and HEATR2). For the genes in this new proposal there is therefore the potential for eventual impact for (1) PCD sufferers and their families (2) The PCD Family Support Group charity (3) Clinicians and other medical staff associated with PCD detection and management (in the UK, the national diagnostic centres for PCD) (4) Clinical researchers investigating the aetiology of PCD and its possible amelioration. We envisage the impact to be (1) facilitate the discovery of new genetic mutations in PCD (2) the potential to improve timely diagnosis of PCD, improving clinical outcome. Rare diseases like PCD don't attract much funding and Drosophila research provides a cost-effective way of impacting such diseases.

In the long term, chaperone pathways are good candidates for routes to therapy as they can be targeted for small molecule modulation (e.g. increasing HSR, decreasing proteasome activity). This is an area that our main collaborator, Pleasantine Mill, is actively exploring in mouse models of PCD.

The ciliary motility defects we investigate also have clear implications for male infertility research, which is frequently due to poor sperm quality, including defective motility.

Other commercial impact

Insects are major pests of agriculture and are vectors of disease. Our research has potential benefits for agrochemical and insect control research. Insect mechanosensory cilium function is a known insecticide target and is a research interest of Syngenta. The ciliary motility genes we investigate are candidate molecular targets of these insecticides. Our collaborator on Drosophila hearing, Joerg Albert (UCL) has strong connections with Syngenta.

Low fertility due to poor sperm quality is a major problem in animal husbandry (e.g. the artificial insemination industry). In many cases sperm immotility is a key problem. Our research will illuminate the molecular mechanisms of sperm motility and provide candidate genes/proteins that could be targeted to improve sperm motility.

NC3Rs impact

Increasingly, cilia research is being conducted in mouse and other protected species. Because of the highly tissue-specific nature of its motile cilia, Drosophila provides an efficient model for ciliary gene discovery and analysis that satisfies the requirement for 'relative replacement' of protected animal species. Our findings directly refine the mouse research of our main collaborator.

Skills/training

The named PDRA on this programme is highly experienced but will benefit from training in new techniques. In the course of this project, she will undertake training in state-of-the-art protein tagging and microscopy techniques (including SNAP tagging and super-resolution microscopy training from our collaborator Pleasantine Mill). This will maintain her employability at the bench, which is her wish. The technician post will likely be filled by a newly qualified graduate, who will gain valuable experience that may well be a stepping stone to a PhD position.

Public engagement

The PI will continue to engage with the Primary Ciliary Dyskinesia Family Support Group (talk and demonstration given at annual meeting in 2017 and likely again in 2018). The wider public is likely to be interested in this research from several points of view. The use of an insect as a research tool with direct relevance to humans is a fascinating and unexpected concept to many, and it is essential to communicate this as an important aspect of BBSRC's support for NC3Rs.