A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome

Abstract

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2

“…As an example, kidney epithelial cell cilia from patients with ADPKD or from Pkd1 and Pkd2 knockout mice were shown to be elongated, possibly due to altered signalling [43, 44]. To investigate how DLG1 might affect ciliary protein composition, we used an unbiased cilium-targeted proximity labelling approach [24] by taking advantage of previously described IMCD3 cell lines stably expressing a ciliary NPHP3[residues 1-203]-BioID2 fusion protein (hereafter called cilia-BioID2) or BioID2 alone (hereafter called BioID2) [45]. We then knocked out Dlg1 in these lines with the aim of determining how loss of DLG1 affects the ciliary proteome.…”

“…#B4501). The cells were lysed, and samples were prepared for mass spectroscopy according to a previously published BioID2-based proximity labeling protocol [45].…”

“…Mass spectroscopy and data analysis. The samples were analyzed and proteins were identified according to the method described in [45]. For proteomics data analysis, we used a custom in-house R script that replicates the analysis using the Perseus software [94].…”

Loss of KIF13B causes time-dependent changes in ciliary polycystin-2 levels and extracellular vesicle release

“…To observe KIF13B dynamics in the primary cilia of mCCD cells, we used mCCD cells stably expressing mNeonGreen-KIF13B. The primary cilia were visualized either by SiR-Tubulin staining or by stable expression of mCherry-RAB8A (Aslanyan et al, 2023). Cells were grown on a 35 mm glass-bottom dish and serum-starved for 24-72 h. For staining with SiR-tubulin dye (Cytoskeleton, Inc., cat.…”

The Diversified Astrocyte Developmental Programs are Modulated by Primary Ciliary Signaling

“…Future work is needed to pinpoint the upstream non-Shh signaling pathways emanating from primary cilia in astrocytes. With the recent advancements in proximity labeling-based proteomics and ciliary purification techniques [110][111][112] , we anticipate that successful application of such methods on the comprehensive profiling of astrocytic cilia will greatly aid further investigations on how primary cilia sense and convey environmental signals to modulate astrocyte biology, and how this process is perturbed to manifest pathogenic changes in ciliopathies and associated diseases such ID, ASD, and epilepsy.…”