PhD thesisThe primary cilium is a singular microtubule-based cell organelle present on most quiescent eukaryotic cell types and is required for numerous signalling pathways. Primary cilia length and incidence have been reported to be altered by many mechanical and chemical stimuli. Furthermore, a group of diseases termed ciliopathies are associated with genetic disruption to primary cilia. Alterations in primary cilia structure have been associated with modified cell signalling events, of which the hedgehog signalling pathway is the most well established as having a ciliary link. The aim of this thesis project was to identify compounds that regulate primary cilia expression in healthy chondrocytes, and in turn to investigate the effect of altered cilia structure on cilia function. It is envisaged that this will support future work to use cilia modifying compounds as potential ‘ciliotherapies’ for the treatment of established ciliopathies and diseases with a ciliary component. A high throughput screen was optimised and executed to enable the unbiased investigation of the effect of 1,727 compounds on cilia expression and structure. Primary bovine articular chondrocytes were utilised; an ideal cell model for the interrogation of primary cilia structure due to their high expression of flat lying cilia in vitro 2D culture. Compound treatment of a confluent monolayer culture was uniformly performed using liquid handling robotics, followed by cellular fixation and immunofluorescent labelling. Nuclei, F-actin and the cilia axoneme components acetylated-α-tubulin and polyglutamylated tubulin were labelled and imaged with automated 3D confocal microscopy. High content image analysis of the 552,960 images generated in first pass screen collected data on over 60 cellular parameters. The data was stored in a database of compound effect with accompanying z-score matrix. From the full library, 233 (13.5%) compounds were identified as altering either cilia length and/or incidence. Using hierarchical clustering, six phenotypic clusters were found amongst these compounds. A cluster of 68 compounds causing an increase in cilia length with a predominantly ciliary specific effect were validated in cells isolated from two additional animals. The 22 validated compounds were carried forward for investigation in a human chondrocyte cell line. The viability of the 22 candidate compounds was tested in a human chondrocyte cell line, of which 16 compounds were found to maintain cell number. Of the viable compounds, 15 increased primary cilia length in the human cells, replicating the effects seen in bovine. The impact of this cilia elongation on subsequent cilia function, specifically on hedgehog signalling was examined by RT-PCR, with ligand induction of the pathway. Although effects were inconsistent, across all compounds there was a trend towards an inhibition to Gli1 induction with the addition of Ihh ligand. These studies identified a robust group of candidate compounds that induced primary cilia elongation in multiple biological replicates using chondrocytes from both bovine and human sources. Although cilia elongation in healthy cells did not cause a consistent effect on ligand-induced hedgehog signalling, these compounds may have future potential as novel ‘ciliotherapies’ to correct cilia dysfunction
