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. The Stephens lab was the first to define the subunit composition of the dynein-2 motor in humans. The Roberts lab have made major advances in our understanding of its regulation and are now working to define its structure and mechanisms of function using in vitro reconstitution. A notable point is that, unlike the related (and better understood) dynein-1 motor, dynein-2 is asymmetric with two key proteins, WDR34 and WDR60, associating with the main motor. Despite being part of the same motor complex, our new work shows that these two proteins have distinct functions. Using genome engineering of cells, we have shown that knocking out WDR34 blocks the ability of cells to form cilia. In contrast, cells lacking WDR60 can still form cilia. However, while cells normally form a tight diffusion barrier at the base of the cilia to gate entry and exit of proteins and lipids. This "transition zone" makes the cilia functionally separate from the rest of the cell. WDR60 knockout cells for cilia that have an abnormal structure and no longer have a tight diffusion barrier to physically segregated the cilia from the rest of the cell.

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. Our current BBSRC-funded work has developed proteomics approaches that have identified key interacting proteins that seem to direct the assembly and function of dynein-2. Here we propose to explore the molecular basis for the role of WDR34 and WDR60 in building the cilium, forming and then maintaining the ciliary transition zone. Building on other data, which we show in this proposal, we will also define how and where the dynein-2 motor is assembled from its component parts. We will also develop a new area of our work to study the balance between ciliogenesis and cell cycle. These processes are mutually exclusive because the same key cellular organelle, the centrioles, are required for both. In resting cells, the centrioles build the cilium, in cycling cells, they are used to build the mitotic spindle that segregates chromosomes between the two resulting daughters. Our work has shown that dynein-2 binds to a centriole protein called CEP170 that has been shown to work to control microtubule dynamics during entry to and exit from the cell cycle. This suggests close integration of dynein-2 function with cell cycle control providing an exciting new area of investigation.

This is a frontier bioscience project that seeks to understand fundamental processes in cell biology. That said, the formation of cilia, tight control of cilia-based signalling pathways, and the control of entry to and exit from the cell cycle are fundamental to normal health as well as having potential long-term impact on human and animal health. Ciliary signals include those that control early human development as well as others that occur throughout life to control metabolism. Key pharmaceuticals targeting common cancers are also directed against ciliary signalling pathway. A full understanding of the structure and function of cilia is key to a diverse array of fields and has relevance from early human development and throughout life.