Spatiotemporal dissection of protein localization to human primary cilia

Primary cilia are solitary, filamentous membrane protrusions emanating from the surface of most human cells. Most of what we know about their function stems from hereditary diseases that are caused by cilia dysfunction, so-called ciliopathies. Ciliopathy patients have a broad spectrum of phenotypes, underlining that cilia fulfil pleiotropic and tissue-specific functions. However, the molecular mechanisms underlying primary cilia function and cell-type specific roles of primary cilia remain poorly understood. This is mainly due to the lack of tools to reveal the protein composition of individual cilia. We address this challenge with antibody-based, spatial proteomics, and develop a cilia atlas as part of the subcellular section of the Human Protein Atlas (HPA) (https://www.proteinatlas.org/humanproteome/subcellular). We co-stained 1800 validated antibodies from the HPA with markers for nuclei, basal bodies, and primary cilia in three human cell lines of different tissue origin: hTERT-RPE1 cells from the retina, RPTEC/TERT1 cells from the kidney proximal tubules, and the mesenchymal stem cell line ASC52telo from the adipose tissue. We acquired high-resolution confocal 3D images and manually annotated as well as computationally analyzed each image.

Our preliminary analysis revealed approximately 500 proteins to localize to primary cilia, with 200 of these proteins also detected at the basal body. We detected an additional 250 proteins that localized to basal bodies but not to cilia. The identified ciliary proteins highlight primary cilia as cellular antennae: At least 170 of the identified ciliary proteins are known to drive signal transduction and engage, e.g., in Hedgehog, GPCR, Calcium, TGFB, RTK, Rho GTPase, MAPK, or mTOR signaling. Furthermore, we observed a remarkable heterogeneity, both between cell lines and within supposedly identical cell populations. For approximately 75% of the identified ciliary proteins, we observed variable amounts in cilia of the same cell line. Furthermore, we identified only about 20% of the ciliary proteins in cilia of all three cell lines. Beyond that, for about 90% of the ciliary proteins, we observed co-localization to other subcellular compartments, with cytosol, basal body, microtubules of the cell body, and nucleoplasm being the most common other compartments. By computational analysis of the images, we relate the dynamic ciliary protein localization to cellular states.

In conclusion, we reveal new ciliary proteins and demonstrate that the primary cilium is the most dynamic and diverse cellular compartment. Our cilia atlas will pave new avenues for basic and clinical research on cilia and ciliopathies.