The ciliary membrane‐associated proteome reveals actin‐binding proteins as key components of cilia

Abstract

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.

Centriolar satellites are required for efficient ciliogenesis and ciliary content regulation

“…In particular, satellites are required for centrosomal actin nucleation through targeting actin assembly factors to the centrosome . Satellites might also function in other actin‐regulated ciliary processes, such as periciliary vesicle transport to the basal body and cilia length regulation . Moreover, we also found a markedly strong increase at the protein level in neurogenesis‐linked pathways, such as neuronal body and extensions, postsynaptic membrane, and axon guidance.…”

Regulation of ciliary membrane protein trafficking and signalling by kinesin motor proteins

“…4B) cells [291]. In agreement with the ciliary presence of KIF13B, well-known KIF13B interactors such as UTRN and DLG1 were identified in ciliary proteomes in several studies [202,262,311]. The recruitment of KIF13B to the centrosome was partly dependent on physical interaction with the TZ protein NPHP4, which binds to at least two distinct sites within the KIF13B tail region, but KIF13B itself is not concentrated at the TZ [291].…”

“…The listed examples suggest a role for cilia in both canonical and noncanonical WNT pathways, which is further supported by the ciliary/basal body localization of proteins important for both pathways . On the other hand, studies of zebrafish Ift88 mutants and mouse Ift88 , Kif3a or Ift72 mutant embryos or fibroblasts (MEFs) showed that, despite the lack of cilia, these did not display WNT‐associated developmental defects or differences in expression of WNT target genes compared to wild‐type organisms and cells .…”

Aldosterone controls primary cilium length and cell size in renal collecting duct principal cells

“…Defects in the primary cilia are associated with a variety of disorders commonly known as ciliopathies, that exhibit the common feature of kidney cysts . These pathologies include mutations of polycystin‐1 (PC‐1) and polycystin‐2 (PC‐2), which cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases, and fibrocystin which are implicated in autosomal recessive polycystic kidney disease (ARPKD) …”