Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81

Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Science (Impact Factor: 33.61). 08/2013; 341(6149):1009-12. DOI: 10.1126/science.1240985
Source: PubMed


Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in
the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of
IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and
mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue
experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells.


Available from: Philippe Bastin
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    • "During ciliary growth, IFT concentrates soluble tubulin in cilia and thereby promotes elongation of the axonemal microtubules. in vitro interaction between tubulin dimers and IFT74–IFT81 complexes (Bhogaraju et al., 2013). We designed modified GFP–-tubulins in which the E-hook was replaced with the corresponding region of -tubulin, altered to substitute seven glutamate residues with alanine or glutamine, or removed entirely (Table S1). "
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    ABSTRACT: The assembly of the axoneme, the structural scaffold of cilia and flagella, requires translocation of a vast quantity of tubulin into the growing cilium, but the mechanisms that regulate the targeting, quantity, and timing of tubulin transport are largely unknown. In Chlamydomonas, GFP-tagged α-tubulin enters cilia as an intraflagellar transport (IFT) cargo and by diffusion. IFT-based transport of GFP-tubulin is elevated in growing cilia and IFT trains carry more tubulin. Cells possessing both nongrowing and growing cilia selectively target GFP-tubulin into the latter. The preferential delivery of tubulin boosts the concentration of soluble tubulin in the matrix of growing versus steady-state cilia. Cilia length mutants show abnormal kinetics of tubulin transport. We propose that cells regulate the extent of occupancy of IFT trains by tubulin cargoes. During ciliary growth, IFT concentrates soluble tubulin in cilia and thereby promotes elongation of the axonemal microtubules. © 2015 Craft et al.
    The Journal of Cell Biology 01/2015; 208(2). DOI:10.1083/jcb.201409036 · 9.83 Impact Factor
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    • "Cloning, expression, and purification of protein complexes from bacterial cultures Full-length C. reinhardtii IFT genes and corresponding fragments were cloned into pEC vectors and expressed in E. coli BL21(DE3), as reported between IFT46 and ODA16 (Ahmed et al., 2008) as well as IFT81/74 and tubulin (Bhogaraju et al., 2013b), in addition to the indication for the transport of motility-related axonemal proteins by IFT56/TTC26/DYF13 (Ishikawa et al., 2014). The structure of the IFT70/52 complex presented in this report shows several surface patches, which are highly conserved between ciliated species (Fig. S2) and are good candidates for ciliary motor or cargo binding sites. "
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    ABSTRACT: Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330-370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281-329 in a complex that interacts directly with IFT70/IFT52330-381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.
    The Journal of Cell Biology 10/2014; 207(2):269-82. DOI:10.1083/jcb.201408002 · 9.83 Impact Factor
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    • "Epitope-tagged katanin p80 localizes to the basal bodies, where katanin’s microtubule-severing activity could act on cytoplasmic microtubules organized around the basal bodies to generate unprotected, depolymerizing microtubule minus ends, thus increasing the concentration of tubulin dimers in the locale where IFT cargo loading occurs [55]. This locally increased tubulin concentration could result in an increased concentration of free tubulin inside the flagellum [56], bringing it to the higher levels necessary to support the nucleation and assembly of the CP microtubules. In the absence of katanin, intraflagellar free tubulin levels may be high enough to support outer doublet elongation but not CP assembly. "
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    ABSTRACT: Most motile cilia and flagella have nine outer doublet and two central pair (CP) microtubules. Outer doublet microtubules are continuous with the triplet microtubules of the basal body, are templated by the basal body microtubules, and grow by addition of new subunits to their distal ("plus") ends. In contrast, CP microtubules are not continuous with basal body microtubules, raising the question of how these microtubules are assembled and how their polarity is established. CP assembly in Chlamydomonas reinhardtii was analyzed by electron microscopy and wide-field and super-resolution immunofluorescence microscopy. To analyze CP assembly independently from flagellar assembly, the CP-deficient katanin mutants pf15 or pf19 were mated to wild-type cells. HA-tagged tubulin and the CP-specific protein hydin were used as markers to analyze de novo CP assembly inside the formerly mutant flagella. In regenerating flagella, the CP and its projections assemble near the transition zone soon after the onset of outer doublet elongation. During de novo CP assembly in full-length flagella, the nascent CP was first apparent in a subdistal region of the flagellum. The developing CP replaces a fibrous core that fills the axonemal lumen of CP-deficient flagella. The fibrous core contains proteins normally associated with the C1 CP microtubule and proteins involved in intraflagellar transport (IFT). In flagella of the radial spoke-deficient mutant pf14, two pairs of CPs are frequently present with identical correct polarities. The temporal separation of flagellar and CP assembly in dikaryons formed by mating CP-deficient gametes to wild-type gametes revealed that the formation of the CP does not require proximity to the basal body or transition zone, or to the flagellar tip. The observations on pf14 provide further support that the CP self-assembles without a template and eliminate the possibility that CP polarity is established by interaction with axonemal radial spokes. Polarity of the developing CP may be determined by the proximal-to-distal gradient of precursor molecules. IFT proteins accumulate in flagella of CP mutants; the abnormal distribution of IFT proteins may explain why these flagella are often shorter than normal.
    Cilia 11/2013; 2(1):15. DOI:10.1186/2046-2530-2-15
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