The role of dynein-2 in building a functional cilium.

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

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.

Technical Summary

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 of cilia biology. Cilia are of great 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. Dynein-2 is a microtubule motor involved in the formation and function of primary cilia. All cells in the body project a primary cilium for their surface and the function of this organelle is essential for normal human development. We were the first to describe the subunit composition of the human dynein-2 complex, the first characterisation of this important motor from any metazoan system. Our more recent work has defined a function for this motor in formation of the axoneme that forms the core of the cilium as well as in building and/or maintaining the ciliary transition zone. This structure acts as a gate to control diffusion of both soluble and membrane proteins into and out of the cilium, and is critical to cilia function to establish and maintain its identity as a signalling hub distinct from the bulk cytoplasm of the cell. Here, we propose to use a combination of in vitro biochemistry, advanced cell imaging, and proteomics to define of the fundamental role of dynein-2 in the formation of cilia, in establishing and maintaining compartment identity, and in ensuring the fidelity of cilia-based signalling. We will also define the location and mechanism of assembly of dynein-2 and explore a new link that we have identified between these processes and the antagonism between ciliogenesis and cell cycle entry.

Planned Impact

There is great interest in the possibility to subvert existing cellular pathways for therapeutic benefit. The dysfunction of these pathways is either a direct or underlying feature of many human diseases. Many 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. 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 clinical genetics and the pharmaceutical industry.

Who might benefit and how?
Clinicians - Ciliopathies are a cohort of diseases that affect 1 in 1000 people. Understanding the core biology of their formation and function is central to a good understanding of the role cilia play in development, disease, and ongoing health. Our work present opportunities to engage with clinical colleagues in terms of diagnosis of "orphan" ciliopathies as well as in exploring the potential to modulate cilia function for improved outcomes. Drugs targeting ciliary signalling (notably the hedgehog signalling pathway) are approved for a variety of cancers making our work of interest to oncologists.
Industry - Cilia sit at a nexus between signalling in the context of normal healthy tissue biology and the onset and progression of cancer. As mentioned above, some cilia-specific signalling pathways such as sonic hedgehog have already been targeted successfully for anti-cancer therapies. Dynein-2 plays a direct role in signal transduction within this pathway presenting an opportunity for direct engagement with those targeting cilia-related cancers such as basal cell carcinoma. Furthermore, there is great interest in control of ciliary pathways that have been linked directly through monogenic disorders linked to obesity. In addition, our recent BBSRC-funded work (ref 5 in the proposal) has triggered interest in licensing reagents generated during the project.
The general public - In addition to the broad benefits that understanding fundamental bioscience brings in the longer term (32x gross value added per public spend), this work addresses directly key areas of health that have the potential to impact both on acute genetic diseases as well as long term health of the general population. Cilia control key aspects of signalling during embryonic development but also throughout life. Key research into their role in tissue repair and regeneration presents one opportunity here to build on our fundamental discovery science.
Bioscience researchers - This project 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 including in intellectual property management, clinical trials, and research policy and management. 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.

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