Abstract
Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.
Highlights
The cilium is a conserved organelle, inferred to have existed in the last eukaryotic common ancestor (LECA) and present in most extant protists, as well as all multicellular animals
Our work provides a novel approach to finding new ciliary proteins, and uncovers a functional association between the BBS complex (BBSome), intraflagellar transport (IFT) and the orthologue of the cone-rod dystrophy protein, RAB28
We hypothesised that ciliogenesis genes are highly expressed during this time period and distinguishable from genes required for general neuronal formation and development, which are expressed during a broader time window (Fig 1A)
Summary
The cilium is a conserved organelle, inferred to have existed in the last eukaryotic common ancestor (LECA) and present in most extant protists, as well as all multicellular animals. Cilium dysfunction in humans is associated with a growing number of so-called ciliopathies that affect virtually all physiological and developmental functions [3]. The canonical cilium of 9 doublet microtubules (MTs) extends from a mother centriole-derived basal body, which connects via distal appendages (transition fibers) to the plasma membrane. The proximal-most 0.2–1.0 μm of the axoneme, called the transition zone, functions in early ciliogenesis, and together with basal body structures provides a permeability barrier that separates the ciliary cytosol and membrane from the cell body [6,7,8]. Additional subregions include the inversin and distal tip compartments, as well as the ciliary pocket, which is a depression of the periciliary membrane where the basal body is rooted [5]. Many ciliopathy proteins and associated complexes localise to particular ciliary subcompartments, where they conduct subdomain-specific functions [5,9]
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