Biology subjects

Junger, K.

Publications and source records attributed to Junger, K..

3 recordsLinked to original sources

bioRxiv2023

DLG1 functions upstream of SDCCAG3 and IFT20 to control targeting of polycystin-2 to the primary cilium

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in 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 a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

cell biology↗

bioRxiv2023

Ciliopathy-associated missense mutations in IFT140 are hypomorphic and have edgetic effects on protein interaction networks

The mechanisms underlying recessive Mendelian diseases and the interplay between genotype and phenotype still need to be better understood. It is therefore necessary to characterise the functional effects of missense mutations at the protein level. Here we focus on missense mutations in the intraflagellar transport protein IFT140, which forms part of the IFT complex A (IFT-A), a crucial component of the ciliary machinery. Mutations in IFT140 can cause a vast spectrum of diseases belonging to the group of ciliopathies, reaching from isolated retinal dystrophy to severe skeletal abnormalities and multi-organ diseases such as Mainzer-Saldino and Jeune syndrome. We hypothesise that missense mutations in IFT140 are hypomorphic leading to quantitative effects on a subset of protein-protein interactions. This may affect complex stability as well as perturbations of protein interaction networks. In this work we assessed how 24 missense mutations in IFT140 affect interactions with other IFT and effector proteins using affinity purification coupled to mass spectrometry. Our data reveals that several mutations in IFT140 are hypomorphic and disrupt the stability of the IFT-A complex to varying degrees in a quantitative way. Allelic combination and the degree of IFT-A complex disruption in analysed missense mutations correlates with the severity of the observed phenotype in a subset of patients. In addition, we show that a distinct subset of mutations in IFT140 shows edgetic effects by disrupting specific PPIs rather than causing a total loss of IFT-A binding. This is the case e.g. with the disease-associated protein TULP3 which is involved in cilia-dependent sonic hedgehog signalling.

cell biology↗

bioRxiv2023

Probing the sub-cellular mechanisms of LCA5-Leber Congenital Amaurosis and associated gene therapy with expansion microscopy

Leber Congenital Amaurosis (LCA) is a group of Inherited Retinal Diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with Ultrastructure Expansion Microscopy (U-ExM), we revealed the structural and molecular defects underlying LCA type 5 (LCA5) with unprecedented resolution. We showed that LCA5-encoded lebercilin, together with Retinitis Pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrated that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced level of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, we probed the LCA5 gene augmentation therapy strategy using U-ExM by monitoring its subcellular outcome. We found that, expression of LCA5 partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, as well as photoreceptor survival.

molecular biology↗