Generation of stable transgenic hTERT-RPE1 cell lines for imaging analysis of cilia tip proteins at high temporal and spatial resolution

Abstract

The primary cilium is a signaling organelle present on the cell surface of most eukaryotic cells. The organelle integrates several signaling pathways important for biological processes such as tissue homeostasis and embryonic development. Disruption of the primary cilium structure and/or function leads to a wide range of developmental diseases termed ciliopathies. Joubert syndrome is an autosomal recessive ciliopathy with mutations found in several ciliary protein encoding genes, including CSPP1 and CEP104. CSPP-L (predominant isoform of CSPP1) and CEP104 interact biochemically and are involved in cilia formation, but their ciliary function is not clarified at molecular level. In the present study, hTERT-RPE1 cell lines were generated that stably express mNeonGreen-CSPP-L or mNeonGreen-CEP104, respectively, at levels not interfering with cell cycle progression or cilia formation. The mNeonGreen-CEP104 cell line is of particular importance since immunofluorescence compatible CEP104 specific antibodies are no longer commercially available. Study of the ciliary localization of CSPP-L and CEP104 fusion proteins by 3D-Structured Illumination Microscopy resolved previously undetermined structural details of CEP104 and CSPP-L at the centrosome and the ciliary tip, and identified co-localization of endogenous CSPP-L and mNeonGreen-CEP104 at the cilia tip. Ciliary entry of mNeonGreen-CEP104 was found to be CSPP-L independent, supporting the hypothesis that intra-ciliary interaction of CEP104 and CSPP-L is required for formation of cilia of normal length. Finally, this thesis lays foundation for advanced live cell imaging studies of the potential dynamic behavior of these important proteins at the centrosome and the cilia tip.