Component Of Intraflagellar Transport Complex Research Articles

High expression of TTC21A predicts unfavorable prognosis and immune infiltrates in clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is the most common pathological type of renal cell carcinoma. Tetratricopeptide repeat domain 21A (TTC21A), known as a component of intraflagellar transport complex A which is essential for the function of cilia, However, the role of TTC21A remains unclear in ccRCC. For the first time, we explore the role and potential mechanism of TTC21A in ccRCC based on multiple databases. Methods: TTC21A expression across all TCGA tumor was analyzed via Tumor Immune Estimation Resource (TIMER) site. The correlation between TTC21A and clinicopathologic characteristics of ccRCC was analyzed with TCGA database. The diagnostic and prognostic value of TTC21A was evaluated by receiver operation characteristic curve, Kaplan-Meier plotter and Cox regression respectively. Moreover, functional enrichment analysis of TTC21A and the co-expression genes were performed by Gene Set Enrichment Analysis. The correlation of TTC21A and immune infiltration were evaluated by single sample Gene Set Enrichment Analysis. Results: Pan-cancer analysis indicated that TTC21A was highly expressed in ccRCC and other cancer. In addition, elevated expression of TTC21A was associated with worse overall survival in ccRCC patients. Functional enrichment analysis showed that TTC21A and the co-expressed genes enriched in glucose metabolism and energy metabolism. Moreover, TTC21A expression was associated with infiltrating levels of dendritic cell, nature killer cell and other immune marker sets. Conclusion: The results of analysis indicate that expression of TTC21A is associated with poor prognosis and immune infiltrating in ccRCC, which suggested TTC21A might be used as a potential predictor and target of treatment in ccRCC.

Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46) and preparation of polyclonal antibody

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.

Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity.

The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3

The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

Biochemical analysis of PIFTC3, the Trypanosoma brucei orthologue of nematode DYF‐13, reveals interactions with established and putative intraflagellar transport components

DYF-13, originally identified in Caenorhabditis elegans within a collection of dye-filling chemosensory mutants, is one of several proteins that have been classified as putatively involved in intraflagellar transport (IFT), the bidirectional movement of protein complexes along cilia and flagella and specifically in anterograde IFT. Although genetic studies have highlighted a fundamental role of DYF-13 in nematode sensory cilium and trypanosome flagellum biogenesis, biochemical studies on DYF-13 have lagged behind. Here, we show that in Trypanosoma brucei the orthologue to DYF-13, PIFTC3, participates in a macromolecular complex of approximately 660 kDa. Mass spectroscopy of affinity-purified PIFTC3 revealed several components of IFT complex B as well as orthologues of putative IFT factors DYF-1, DYF-3, DYF-11/Elipsa and IFTA-2. DYF-11 was further analysed and shown to be concentrated near the basal bodies and in the flagellum, and to be required for flagellum elongation. In addition, by coimmunoprecipitation we detected an interaction between DYF-13 and IFT122, a component of IFT complex A, which is required for retrograde transport. Thus, our biochemical analysis supports the model, proposed by genetic analysis in C. elegans, that the trypanosome orthologue of DYF-13 plays a central role in the IFT mechanism.

HA‐tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B

Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Chlamydomonas IFT172 Is Encoded by FLA11, Interacts with CrEB1, and Regulates IFT at the Flagellar Tip

The transport of flagellar precursors and removal of turnover products from the flagellar tip is mediated by intraflagellar transport (IFT) , which is essential for both flagellar assembly and maintenance . Large groups of IFT particles are moved from the flagellar base to the tip by kinesin-2, and smaller groups are returned to the base by cytoplasmic dynein 1b. The IFT particles are composed of two protein complexes, A and B, comprising approximately 16-18 polypeptides. How cargo is unloaded from IFT particles, turnover products loaded, and active IFT motors exchanged at the tip is unknown. We previously showed that the Chlamydomonas microtubule end binding protein 1 (CrEB1) localizes to the flagellar tip and is depleted from the tips of the temperature-sensitive (ts) mutant fla11ts . We demonstrate here that FLA11 encodes IFT protein 172, a component of IFT complex B, and show that IFT172 interacts with CrEB1. Because fla11ts cells are defective in IFT particle turnaround at the tip, our results indicate that IFT172 is involved in regulating the transition between anterograde and retrograde IFT at the tip, perhaps by a mechanism involving CrEB1. Therefore, IFT172 is involved in the control of flagellar assembly/disassembly at the tip.