The dynein-2 microtubule motor

Nearly all cells in the human body build a primary cilium, an antenna-like structure that emerges from the surface of nearly all human cells. Defects in the formation and/or function of the cilium lead to a cohort of human diseases known as the ciliopathies. This project does not seek to define the molecular basis of disease but instead is solely directed at a fundamental understanding o cilia biology. Cilia are of particular importance for developmental signalling and therefore, dynein-2 underpins the normal function of all cells in the body. Cilia are built around a microtubule rich structure along which molecules are transported by motor proteins. Considerable work has now shown that the cytoplasmic dynein-2 motor complex is essential for normal human development because of its function in both directing molecules into cilia as well as controlling their movement along cilia. Dynein-2 is distantly related to dynein-1, a complex that is relatively well understood in terms of function and particularly well in terms of its composition. These two "cytoplasmic" dyneins move large molecular complexes around inside cells and are distinct from the "axonemal" dyneins that drive beating of cilia and flagella. In contrast to the axonemal dyneins and to cytoplasmic dynein-1, until very recently we have known little about the individual proteins that go to make up the dynein-2 machine. Here, I propose to build on our work that has, for the first time, defined the biochemical composition of the human dynein-2 complex. Building on a strong track record of work linking membrane trafficking and microtubule motors, I propose to use a combination of biochemistry, advanced cell imaging, and selective proteomics to provide a clear definition of the fundamental biology of dynein-2 in cells.

Primary cilia project from the surface of nearly all human cells to serve as signalling platforms. The dynein-2 microtubule motor provides a fundamental link between microtubule motor function, protein trafficking, and cilia function because of its function in driving intraflagellar transport (IFT) within cilia. We have recently defined the subunit composition of the dynein-2 motor. Most notable among our findings are that the intermediate chain subunits of this motor, unlike the related dynein-1 motor, are asymmetric with both WDR34 and WDR60 being associated with the same heavy chain subunit. We also identified a dynein light chain, TCTEX1D2, which we showed to be associated with dynein-2 but not dynein-1. Importantly, we also showed that dynein-2 does not associate with the known canonical dynein-1 regulators dynactin, LIS1, or Nudel. Consequently while we know the bare composition of the motor, we know little about the mechanisms that control its assembly, activity, or coupling to cargo. Our proposal seeks to provide a complete picture of the molecular interactions of the dynein-2 complex using a combination of molecular cell biology approaches including advanced microscopy and proteomics. We will define the interactome of dynein-2 using proteomics, focussing on the two intermediate chain subunits WDR34 and WDR60. We will further explore the role of the unique dynein-2 light chain TCTEX1D2 using CRISPR-mediate genome editing and also generate cell lines null for both WDR34 and WDR60 to provide a blank canvas for analysis of the function of these genes using site-directed mutagenesis. We will develop a selective proteomics method to define the composition of cilia and use this to probe the role of dynein-2 in this key process. Our work seeks to address the fundamental cell biology of this intriguing motor and shed light on its role in normal cell function and human development.

There are key aspects within the project that have potential to be of use in the pharmaceutical and related industries. There is great interest in the possibility to subvert existing cellular pathways for therapeutic benefit. In addition, the dysfunction of these pathways is either a direct or underlying feature of many human diseases. In recent years, several human congenital diseases have been determined to be caused by mutations in genes encoding the cilia machinery. These diseases span a range of physiological steps from skeletal development to kidney function. This highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. Thus, the potential impact of our work is without question. While it is always more complex to define the way in which and timescales for such impacts might occur, we can develop such lines through our impact plan. Through informing our basic understanding of a critical cellular process, it is most likely our work will inform long term projects in other fields including the pharmaceutical industry.

In this way, potential applications of this work are identified from within the department (through regular discussion with our Impact lead and industrial liaisons) as well as by continuing discussions with our Research and Enterprise Department. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders such as MRC when appropriate. As with all of our projects, this one includes considerable opportunity to train the researchers involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes and the extensive network of University schemes to benefit the training and development of research staff (Bristol is at the forefront of research staff development). I have a good track record in facilitating the placement of staff in areas outside our core research activity. For example, a previous postdoc in the lab undertook a period of flexible working in order to shadow some of our Research and Enterprise team and subsequently undertook a part-time course in intellectual property management. She has now moved to such a position with a major company working in this area. This demonstrates that the environment provided by my own lab a well as the University as a whole is highly conducive to career development of our staff beyond academic, basic science research alone and thus contributes to the economic development of the nation. Our projects are also very data intensive - notably from imaging work - and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life.

This work will lead to significant image data that is readily used in both public understanding of a science and artistic arenas. Examples include local exhibitions and promotions. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore is likely to contribute to local exhibitions or displays as has been the case with previous work from our lab and others within our School.