Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT‐B complex consists of 9–10 stably associated core subunits and six “peripheral” subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six “peripheral” IFT‐B subunits of Chlamydomonas reinhardtii as recombinant proteins and show that they form a stable complex independently of the IFT‐B core. We suggest a nomenclature of IFT‐B1 (core) and IFT‐B2 (peripheral) for the two IFT‐B subcomplexes. We demonstrate that IFT88, together with the N‐terminal domain of IFT52, is necessary to bridge the interaction between IFT‐B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT‐B1/IFT‐B2 complex formation. Furthermore, we show that of the three IFT‐B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ‐tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface‐exposed residues.     

Intraflagellar transport and cilia-dependent diseases

Intraflagellar transport involves the movement of large protein particles along ciliary microtubules and is required for the assembly and maintenance of eukaryotic cilia and flagella. Intraflagellar-transport defects in the mouse cause a range of diseases including polycystic kidney disease, retinal degeneration and the laterality abnormality situs inversus, highlighting the important role that motile, sensory and primary cilia play in vertebrates.     

The intraflagellar transport machinery of Chlamydomonas reinhardtii

First discovered in the green alga, Chlamydomonas , intraflagellar transport (IFT) is the bidirectional movement of protein particles along the length of eukaryotic cilia and flagella. Composed of ∼16 different proteins, IFT particles are moved out to the distal tip of the organelle by kinesin-II and are brought back to the cell body by cytoplasmic dynein 1b. Mutant analysis of the IFT motor and particle proteins using diverse organisms has revealed a conserved and essential role for IFT in the assembly and maintenance of cilia and flagella. IFT is thought to mediate this assembly through the delivery of axonemal precursors out to the distal tip of the growing organelle. Consistent with this model, the IFT particle proteins are rich in protein–protein binding motifs, suggesting that the particles may act as scaffolds for the binding of multiple cargoes. With most of the IFT proteins now identified at the level of the gene, this review will briefly examine both the structure and function of the IFT machinery of Chlamydomonas reinhardtii .     

Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.     

Crystal structure of the intraflagellar transport complex 25/27

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub‐complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure‐guided mutagenesis. IFT27 displays the fold of Rab‐like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase‐activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP‐binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high‐resolution structural understanding of the IFT complex.     

Rab34 GTPase mediates ciliary membrane formation in the intracellular ciliogenesis pathway

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ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

The role of primary cilia in the development and disease of the retina

The normal development and function of photoreceptors is essential for eye health and visual acuity in vertebrates. Mutations in genes encoding proteins involved in photoreceptor development and function are associated with a suite of inherited retinal dystrophies, often as part of complex multi-organ syndromic conditions. In this review, we focus on the role of the photoreceptor outer segment, a highly modified and specialized primary cilium, in retinal health and disease. We discuss the many defects in the structure and function of the photoreceptor primary cilium that can cause a class of inherited conditions known as ciliopathies, often characterized by retinal dystrophy and degeneration, and highlight the recent insights into disease mechanisms.     

The role of primary cilia in the development and disease of the retina

The normal development and function of photoreceptors is essential for eye health and visual acuity in vertebrates. Mutations in genes encoding proteins involved in photoreceptor development and function are associated with a suite of inherited retinal dystrophies, often as part of complex multi-organ syndromic conditions. In this review, we focus on the role of the photoreceptor outer segment, a highly modified and specialized primary cilium, in retinal health and disease. We discuss the many defects in the structure and function of the photoreceptor primary cilium that can cause a class of inherited conditions known as ciliopathies, often characterized by retinal dystrophy and degeneration, and highlight the recent insights into disease mechanisms.     

Ciliary Length Sensing Regulates IFT Entry via Changes in FLA8/KIF3B Phosphorylation to Control Ciliary Assembly

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TPPP orthologs are ciliary proteins

Tubulin polymerization promoting protein, (TPPP/p25), was identified as a brain-specific protein. The potential function of this protein resembled that of MAPs. It is mainly expressed in oligodendrocytes; however, immunopositivity was also detected in glial and neuronal inclusions in synucleinopathies. Here, we show that TPPP gene(s) are conserved in the genomes of ciliated organisms, but are lacking from the nonciliated ones. This recognition is based upon homologous gene sequence analysis, in silico comparative genomic studies, bioinformatic search and experimental evidence. Cilia (flagella) are microtubule-based cellular extensions of sensory and/or motile function. TPPP orthologs are among the only 16 genes that can be found in all ciliated organisms, suggesting that TPPP orthologs may be associated with a basic function of cilia.     

Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control.

A central question in cell biology is how cells determine the size of their organelles. Flagellar length control is a convenient system for studying organelle size regulation. Mechanistic models proposed for flagellar length regulation have been constrained by the assumption that flagella are static structures once they are assembled. However, recent work has shown that flagella are dynamic and are constantly turning over. We have determined that this turnover occurs at the flagellar tips, and that the assembly portion of the turnover is mediated by intraflagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the flagellar tips and causes the flagella to resorb. These results lead to a simple steady-state model for flagellar length regulation by which a balance of assembly and disassembly can effectively regulate flagellar length.     

Architecture and function of IFT complex proteins in ciliogenesis

Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.