Schermer, Bernhard | Liebau, Max C - C 14

The overarching aim of our project is to gain novel insights in the disease mechanisms of a large heterogeneous group of severe human diseases termed ciliopathies.

While individual ciliopathies are rare conditions, they collectively struck a large number of individuals. We expect our studies to challenge and expand several current pathophysiological concepts in the large field of ciliopathy research.

This will be important for many medical disciplines since ciliopathies manifest in multiple organs (e.g. kidney, brain, eyes, pancreas, liver). Ultimately, we hope to provide novel key mechanisms and potential therapeutic targets to the pathogenesis of ciliopathies.

Groundbreaking work of the last two decades has revealed that mutations in genes encoding for proteins localized to primary cilia are causative for a large number of different human diseases, nowadays subsumed as a group termed ciliopathies.

A hallmark of most ciliopathies is the development of cystic kidneys caused by altered function and proliferation of epithelial cells. Multiple other tissues can also be severely affected. Beyond hereditary ciliopathies, there is accumulating evidence that cilia may be involved in the pathogenesis of various acquired diseases including diabetes and cancer.

Primary cilia are sensory organelles that, like antennae, project from the surface of virtually all mammalian cells explaining the broad spectrum of defects and clinical symptoms observed in ciliopathies. To exert their signaling functions, cilia are covered by a specialized compartment of the plasma membrane and the entry of proteins into the ciliary compartment and the ciliary membrane is strictly regulated.

While basic mechanisms of ciliogenesis, ciliary disassembly, ciliary targeting, intra-flagellar transport and ‘classical’ ciliary signaling such as Hedgehog or WNT signaling have been extensively studied in the recent past, the exact molecular alterations of primary cilia in human ciliopathies have not been identified so far due to technical limitations. Since the vast majority of ciliopathy-causing mutations do not result in the loss of ciliogenesis, a detailed understanding of the subtle and largely unknown alterations of cilia in ciliopathies is urgently needed to decipher the mechanisms of disease.

To break down technical limitations in studying ciliary protein composition we fused the engineered peroxidase APEX2 to a ciliary targeting motiv and used proximity labeling and pulldowns followed by MS/MS analyses to explore the ciliary membrane associated proteome.

This approach revealed numerous novel ciliary proteins unexpectedly including actin-binding proteins and even more unforeseen families of proteins.

The overarching goal of the at hand project is to use and expand this technology to get a deeper understanding of the molecular composition of cilia and their alterations in ciliopathies both in patient derived cells and model organisms.

To overcome limitations in the analysis of the ciliary proteome we used APEX-based proximity labeling¹ to biotinylate and pull down ciliary membrane associated proteins for subsequent MS/MS analysis (‘ciliary membrane APEX’ (cmAPEX);²). With this approach we identified numerous novel ciliary proteins in renal epithelial cells. Among those we found actin binding proteins to be very prominent. Following up on this, we could demonstrate that myosin 5a is essential for ciliogenesis by using knockout (KO) cell lines generated with CRISPR/Cas9. This function has been confirmed independently by another lab.³

We recently used the same experimental approach to study ciliary disassembly. In brief, we induced ciliogenesis by serum starvation for 48 h and stimulated ciliary disassembly by serum addition. The ciliary proteome was determined at 45 min, 2 h and 8 h after serum addition. Interestingly, within these datasets, a number of proteins related to unexpected biological processes were detectable with an increased abundance. We are currently following up on these hits and hope to obtain a deeper understanding of the interplay of cilia with other processes and structures of cellular biology. In summary, ciliary proximity labeling allows the comprehensively analysis of the ciliary proteome with very high sensitivity and provided some more evidence on a potential role of cilia in cellular biology.

1. Rhee H-W, Zou P, Udeshi ND, et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 2013;339(6125):1328–31.
2. Kohli P, Höhne M, Jüngst C, et al. The ciliary membrane-associated proteome reveals actin-binding proteins as key components of cilia. EMBO Rep 2017;18(9):1521–35.
3. Wu C-T, Chen H-Y, Tang TK. Myosin-Va is required for preciliary vesicle transportation to the mother centriole during ciliogenesis. Nat Cell Biol 2018;20(2):175–85.