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DLG1 Functions Upstream of SDCCAG3 and IFT20 to Control Ciliary Targeting of Polycystin-2
Background: DLG1 is a multifaceted protein that belongs to the membrane-associated guanylate kinase (MAGUK) multidomain scaffolding protein family. As a core component of the Scribble polarity complex located at the basolateral membrane below the adherens junctions, its primary function is to establish apical-basal polarity in epithelial cells [1]. Furthermore, several studies have implicated DLG1 in various exocytotic and endocytotic processes, e.g., in neurons where DLG1 regulates neuronal receptor localization and clustering [2]. Interestingly, three independent studies found DLG1 in the ciliary proteome of cultured mouse kidney epithelial cells [3-4] and photoreceptor outer segments [5], indicating that DLG1 is localizing to the primary cilium. However, cilia-related functions for DLG1 have so far not been described. On a physiological level, it has been reported that ablation of Dlg1 in mice leads to severe kidney phenotypes [6-8]. Similarly, in humans, DLG1 has been identified as a susceptibility gene for congenital anomalies of the kidney and urinary tract (CAKUT) [9]. Consistently, a patient with CAKUT was discovered to possess a missense mutation in DLG1 (p.T489R) [10]. However, it remains uncertain if some of the kidney phenotypes observed in mice and humans with DLG1 mutations are associated with ciliary defects.
Aims: This study aimed to investigate a potential ciliary function for DLG1 in cultured mouse kidney epithelial cells, as well as in vivo.
Methods: Culture and transfection of mouse inner medullary collecting duct 3 cells (IMCD3) and mouse cortical collecting duct (mCCD) cells; gene knock-out and generation of stable rescue and mutant cell lines; BioID2-based proximity labeling and mass spectrometry; immunohistochemistry analysis of kidney sections from WT and Pax3Cre-Dlg1F/F mice; immunofluorescence microscopy (IFM) and automated image analysis; co immunoprecipitation; SDS-PAGE and western blotting; AlphaFold modeling.
Results: Conditional knockout of Dlg1 in mouse kidneys caused ciliary elongation and cystogenesis. Similarly, loss of Dlg1 in cultured mCCD cells led to ciliary elongation, and this phenotype was rescued by exogenously expresses wild-type DLG1 but not the CAKUT-associated DLG1T489R variant. Further experiments using cell-based proximity labeling proteomics and IFM analysis showed that loss of DLG1 resulted in alterations to the ciliary proteome. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) were reduced in the cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not the DLG1T489R variant. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1.
Conclusion: We discovered that DLG1 plays a key role in regulating the length and composition of primary cilia in kidney epithelial cells, both in cultured cells and in vivo. Specifically, DLG1 mediates the ciliary targeting of several proteins, including SDCCAG3, IFT20, and PC2. Furthermore, ourfindings suggest that ciliary dysfunction of the DLG1T489R variant may contribute to CAKUT [11].
Funding: This work was supported by grants NNF18SA0032928 and NNF22OC0080406 from the Novo Nordisk Foundation (LBP, SFP), grant 2032-00115B and 3103-00177B from the Independent Research Fund Denmark (LBP, STC), grant R01- DK108005 from the National Institute of Diabetes and Digestive and Kidney Diseases (MRM), Innovation grant 20OI174 from the Dutch Kidney Foundation (RR), the European Union's Horizon 2020 research and innovation program Marie Sklodowska-Curie Innovative Training Networks (ITN) grant 861329 (RR, RBR, KB, STC, LBP), and the Wellcome Trust grant 201585/B/18/Z (GDD, RBR).
Speakers
Csenge K. Rezi