Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways

Abstract

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

The transcriptional signature associated with human motile cilia

“…Apart from being a possible false negative, this discrepancy could be indicative of the diversity of ciliary components in eukaryotes (Nevers et al, 2017). A recent study using evolutionary proteomics has predicted MYCBPAP to be associated with cilia, and ISH of the FAM183A orthologue has shown to positively stain motile ciliated tissue in Xenopus laevis (Beckers et al, 2018, Sigg et al, 2017) . Additionally, EFCAB6 has previously been associated with sperm motility in human through proteomics (Amaral et al, 2014).…”

FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

“…Although several studies have identified FAP57/WDR65 as a conserved component of motile cilia and flagella (Broadhead et al, 2006;McClintock et al, 2008;Arnaiz et al, 2010;Sigg et al, 2017;Blackburn et al, 2017), functional studies on fap57/wdr65 mutations are very limited. One report identified a missense mutation in WDR65 in a patient with Van der Woude syndrome, a cleft palate disorder, but this work did not establish a clear connection between the mutation and the cleft palate defects (Rorick et al, 2011).…”

“…Given the complexity of the microtubule-based 9+2 axonemal structure, motile ciliopathies are often under-diagnosed because of their genetic heterogeneity and multisystem variability (Werner et al, 2015). Yet many proteins of the ciliary axoneme are highly conserved (Li et al, 2004;Pazour et al, 2005;Albee et al, 2013), and so study of motile cilia in model organisms has provided insight into numerous genes and gene products potentially associated with PCD and other ciliopathies (Mitchison and Valente, 2017;Sigg et al, 2017).…”

“…Given the complexity of the microtubule-based 9+2 axonemal structure, motile ciliopathies are often under-diagnosed because of their genetic heterogeneity and multisystem variability (Werner et al, 2015). Yet many proteins of the ciliary axoneme are highly conserved (Li et al, 2004;Pazour et al, 2005;Albee et al, 2013), and so study of motile cilia in model organisms has provided insight into numerous genes and gene products potentially associated with PCD and other ciliopathies (Mitchison and Valente, 2017;Sigg et al, 2017).Genomic and proteomic strategies have identified more than 600 proteins as structural components of the axoneme, and many other proteins contribute to the pre-assembly of axonemal complexes in the cytoplasm, their delivery to the basal body region, and their transport through the transition zone and into the ciliary compartment (reviewed in van Dam et al, 2019). Advances in high resolution imaging in combination with the ordered and repetitive nature of the axoneme structure have provided insight into the location of several axonemal complexes (Mizuno et al, 2012), but still only a third or so of the ciliary proteins have been clearly correlated with a specific structure.…”

The conserved ancestral signaling pathway from cilium to nucleus

“…It acts as a sensory antenna of these cells (Singla and Reiter, 2006;Satir et al, 2010;Anvarian et al, 2019) and is the basis of sensory organs, such as the vertebrate eye, and certain insect and invertebrate sensilla. It is closely related to motile 9+2 cilia, which are widely distributed from protists to man and also have sensory functions (Sigg et al, 2017). Mutations in ciliary proteins have been linked to human diseases including polycystic kidney disease (PKD) and primary ciliary dyskinesia (PCD), now collectively called ciliopathies (Fliegauf et al, 2007;Waters and Beales, 2011).…”