The Golgi apparatus as an initiator of ciliogenesis

The ability of cells to sense and respond to the environment is critical at all stages of life. This is perhaps most important during development as cells differentiate into specific tissue types. Most cells in the human body extend a primary cilium that acts as an antenna to sense and respond to the extracellular environment. Primary cilia are required for proper developmental signalling and any defect in a cells ability to form a cilium causes serious developmental defects. These diseases, collectively known as the ciliopathies, include specific syndromes such as Meckel-Gruber and Bardet-Biedel Syndrome as well as polycystic kidney disease and short rib polydactyly. We are beginning to understand the causes of these diseases by defining the causative mutations in single genes. A key to this is to ensure that we fully understand the way in which these primary cilia are built and maintained. Cilia are extensions of a specialised set of microtubule filaments called the axoneme that are surrounded by a high specialised membrane. This process requires a close cooperation of two key cellular elements - the cytoskeleton and the membranes. We have identified a source of the initial membrane that drives the formation of the cilium. For many years we have worked on transport of membranes and proteins through the cell and recently this has led us serendipitously to discover that one of the key proteins that we have studies for many years, giantin, is in fact required for cells to make cilia. We have identified a mechanism by which this occurs which links giantin, a membrane protein of the Golgi apparatus, to dynein-2, one of two of the main motor proteins that drives transport within the cilium. The Golgi is the central organelle of the secretory pathway in all mammalian cells and it is responsible for the modification and sorting of proteins destined for all major cellular organelles. As such it also lies directly adjacent to the centrosome from which the microtubule axoneme extends. Dynein-2 is known to be required for cilium function and mutations in this complex cause a variety of cilia-related disorders in vitro and in patients. We propose that giantin acts through dynein-2 to control the earliest stages of assembly of cilia. Now we wish to use our extensive experience of membrane and microtubule dynamics, in particular live cell imaging, to define this role in detail. We have established collaborations with key labs in the UK, USA, and France that mean we are well placed to drive this work forwards. Our experiments will study the delivery of key components to the newly emerging cilium as well as the organization of membranes around the microtubule axoneme. We expect these experiments to define the role of the Golgi in forming the cilium and lead to new avenues of research in terms of pathways that one might approach to modulate ciliary function as well as identifying candidate genes that might underlie those ciliopathies for which a genetic defect is not yet defined.

The formation and function of primary cilia is critical to the normal development of tissues, organs and ultimately organisms. Defects in these processes cause severe developmental abnormalities. These diseases, collectively known as the ciliopathies, are causes by mutations in genes encoding the machinery that forms and maintains the cilium. These include centrosomal proteins, those that mediate assembly of the ciliary membrane and deliver critical signalling molecules to this highly specialised structure. Key questions remain unanswered about the origin of the membrane that docks adjacent to the mother centriole as it transitions to become a basal body. Subsequent extension of the microtubule axoneme from this basal body is accompanied by significant expansion of the specialised ciliary membrane and delivery of critical signalling molecules that define its subsequent function. Assembly of the cilium and its subsequent function requires the activity of molecular motors that drive translocation of particles along the axoneme. Dynein-2 is centrally involved in this process and considerable genetic data from experimental systems and patients implicate dynein-2 in the initial formation of the cilium itself as well.

Our preliminary data have implicated the Golgi membrane protein giantin in this process. Acting through the dynein-2 motor, our model is that giantin acts to deliver critical components, possibly even the initial membrane itself to the newly forming cilium. Here, we will use a combination of advanced light and electron microscopy approaches, supplemented with biochemical assays, to define whether giantin acts as a hub to recruit dynein-2 during ciliary assembly, the role of giantin and dynein-2 in the targeted delivery of ciliary components, and the relationship to other pathways known to be required for cilium formation. Our work will provide key mechanistic insight into the processes that govern both the formation and function of primary cilia.

There are key aspects within the project that have potential to be of use in the development of technologies within 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 machinery of the cilium. These diseases span a range of physiological steps from skeletal development to erythropoiesis. This highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. It is also anticipated that other diseases that relate to key signalling pathways the operate through primary cilia might be ameliorated by enhancing the efficiency of ciliary function. Dissemination of our findings is the best way to alert those looking to diagnose "orphan ciliopathies" to the potential relevance of GOLGB1 and related genes in ciliopathies. 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 clinical diagnostics and pharmaceutical industries. For example, should we define that our work impacts more directly on cilia-based signalling, it is likely that this work would impact on the field of childhood development. At this point we would engage with those contacts we have in this area to explore possible areas of therapeutic benefit (e.g. the group with whom we made contact with at Cilia 2012). In this way, potential applications of this work are identified from within the department as well as by continuing liaison 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 researcher involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration without public communication and outreach programmes, 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 (MRC-funded) 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 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.