A High-Content Screening Approach to Unveil the Molecular Mechanism of the Ciliary Response to Glutamine

Background: Polycystic Kidney Disease (PKD) is the most common ciliopathy, caused by malfunctioning primary cilia. Primary cilia are microtubule-based organelles at the cell surface that integrate metabolic and signaling cues, though their exact sensory function is unclear. We recently demonstrated that primary cilia sense nutrient availability and adjust the cellular metabolic response. Glutamine deprivation causes cilia elongation, while its supplementation induces shortening due to TCA cycle anaplerosis. We identified asparagine synthetase (ASNS) as a key enzyme; its silencing reduces both anaplerosis and cilia shortening. We aim to identify the full pathway driving this cilia response, believing it will be crucial in PKD. Methods: mIMCD3 stable transfectants with Arl13b-GFP were generated, subcloned, and used to set up an automated quantification of ciliary length in response to glutamine using the ImageXpress Micro Confocal Imaging System by Molecular Devices, scalable to 384 wells. Cells were transfected with key siRNAs for setup studies, and 48 hours later, were analyzed by western blotting or for cilia length. For each well, 16 fields were acquired, measuring over 1,000 cilia per condition, with post-image analysis performed using MetaXpress Custom Modules software. Results: To clarify the molecular mechanism behind ciliary dynamics under stress conditions, we developed a high-content screening method to automatically measure cilia length in response to various stimuli and optimized conditions for silencing known pathway components. According to literature, silencing Ift88, Arl13b, Ccdc41, and Prpf8 induced ciliary shortening, while silencing Asns, Vdac1, Kif2a, and Cep170 led to elongation. The system is now operational. We plan to use a whole genome siRNA library to identify pathway components that drive elongation without glutamine and promote shortening with glutamine replenishment. Conclusion: Understanding primary cilia's role in regulating cellular metabolism is crucial for insights into their functions in health and disease. Dysfunction of primary cilia leads to various ciliopathies, including kidney disorders like PKD. Our data support using a high-content screening platform to identify novel components of the pathway responsible for the ciliary response to glutamine. Funding: Private Foundation Support, Government Support – Non-U.S.