Investigating how Defects in Nuclear Organisation Contribute to Ciliopathies

Abstract

The highly-conserved exocyst complex plays key roles in vesicle transport and establishment of polarity. It predominantly localises to sites of active cell growth. However, there is emerging evidence that the exocyst functions in other cellular contexts. The exocyst serves several functions at primary cilia, which culminate in a strong association between genetic mutations in the exocyst and ciliopathies. However, whilst we know it localises to these structures and performs essential roles, the specific mechanisms by which it contributes to ciliogenesis and ciliary function remain unknown. I hypothesised that the exocyst’s contribution to ciliogenesis extended beyond its role in vesicle trafficking. Here, I use a combination of molecular, cell biology and biochemistry techniques to show that the exocyst is required for ciliogenesis via both the vesicle-dependent and independent routes. I recapitulated two ciliopathy-associated exocyst mutations, EXOC4 p.Q578R and EXOC8 p.E265G, and demonstrate that these are detrimental to the exocyst’s functions in ciliogenesis. In addition, I report novel roles for the exocyst at the nuclear envelope that contribute to ciliogenesis. I show that exocyst subunits localise to the inner face of the nuclear envelope, as well as throughout the nuclear matrix. Here, they are required to mediate the composition of both the nuclear lamina and transmembrane proteins in the nuclear envelope, with (likely) consequential effects for both nuclear morphology and chromatin architecture. As functional links have recently been established between the nuclear envelope and ciliogenesis, I examined whether the roles of the exocyst at these sites were also linked. I report that lamin levels are also altered in other ciliopathy models, and that reduced lamin expression is upstream of the ciliogenesis defect in exocyst-depleted cells. In conclusion, this thesis provides an attractive model to inform future studies, whereby ciliopathy proteins around the nuclear periphery come together to control nuclear envelope composition and lamina organisation, with potential consequences for a plethora of cellular processes.