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High-Content Imaging of Ciliogenesis and Cilia Function, Colin Johnson, Leeds Institute for Molecular Medicine, Leeds

Colin Johnson

Primary cilia are microtubule-based organelles that act as a universal cellular “antennae” in vertebrates to receive and integrate signals from the extracellular environment. They have essential roles in mechanosensation and photosensation, allowing us to hear, see, excrete and reproduce. Defects in cilia cause a range of developmental phenotypes grouped into conditions called ciliopathies, an ever-expanding group of genetic disorders.

We developed a whole-genome small interfering RNA (siRNA)-based reverse genetics screening approach, using the Operetta system, to investigate cilium structure and function. The objective was to identify novel regulators of cilia formation and ciliopathy genes.

The siRNA screen was carried out in the mouse inner medullary collecting duct (mIMCD3) ciliated cell-line, using an siGENOME library containing pools of four siRNA duplexes per gene. A total of 194 genes were identified that significantly affected ciliogenesis and were of particular functional interest. These then underwent further validation in knock down experiments in mIMCD3 and other ciliated cell-lines.

The screen predicted genes that were mutated in ciliopathies or “centriopathies”. Mutations in the validated hit PIBF1/CEP90 were found tocause Joubert syndrome, while mutations in the validated hit C21orf2 cause the ciliopathy, Jeune syndrome. The screen also predicted new functional modules for ciliogenesis. For example, the C21orf2-SPATA7-NEK1 complex was shown to link the pathogenic mechanism of a ciliary chondrodysplasia with retinal dystrophy. Finally, we were able to validate several ciliary G-protein coupled receptors that mediate cilia formation, providing new insights into the regulation of ciliary signalling pathways.

siRNA screen

siRNA screen predicts new functional modules for ciliogenesis: validated ciliary G-protein coupled receptors mediate
ciliogenesis Images courtesy of Dr. Gabrielle Wheway and Dr. Zakia Abdelhamed from the laboratory of CAJ.

In summary, HCS studies can be used to investigate ciliary biological processes and ciliopathy disease mechanisms, to predict new ciliopathy-associated genes and ciliary-related conditions, and to predict novel targets for chemical library screens and therapeutic interventions.

For Research Use Only. Not for Use in Diagnostic Procedures.