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. 2010 Aug 1;21(15):2696-706.
doi: 10.1091/mbc.e10-03-0191. Epub 2010 Jun 9.

Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

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Free PMC article

Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

Zhen-Chuan Fan et al. Mol Biol Cell. .
Free PMC article

Abstract

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1-depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.

Figures

Figure 1.
Figure 1.
IFT70 is a highly conserved protein. (A) Amino acid sequence alignment among the IFT70/DYF-1 othologues from invertebrate and vertebrate species including human (EAX11053), zebrafish (ABV08791), C. reinhardtii (XP_001692406), C. elegans (NP_491494), and Tetrahymena (XP_001033553). (B) The phylogenetic tree of IFT70/DYF-1 proteins. Branch lengths represent evolutionary relatedness.
Figure 2.
Figure 2.
IFT70/CrDYF-1 has a typical localization pattern for IFT proteins, and its entrance into flagella is FLA10 dependent. (A) The wild-type (wt) flagella extract was probed with the affinity-purified α-IFT70/CrDYF-1 antibody with a single band detected. In contrast, preimmune serum does not recognize any specific band. (B) IFT70/CrDYF-1 is localized in the peri-basal body region and flagella. The wt cells were double-labeled with antibodies α-IFT70/CrDYF-1 (green) and α-tubulin (red). The staining with α-tubulin illustrates the position of the two flagella. IFT70/CrDYF-1 is localized primarily in the peri-basal body region as well as in dots along the flagella. The inset shows an enlargement of one of the flagella. (C) The entrance of IFT70/CrDYF-1 into the flagella is FLA10-dependent. Flagellar proteins were extracted from the wt and fla10ts cells after incubation for 50 min at either the permissive temperature (22°C) or the restrictive temperature (32°C), separated on an 8% polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies against IFT70/CrDYF-1 and other IFT complex proteins, as indicated on the right of the Western blots. Equal amount of flagellar proteins were loaded for each sample, as shown by the Coomassie Blue–staining gel in the left panel. The labels A and B represent IFT complexes A and B, respectively.
Figure 3.
Figure 3.
IFT70/CrDYF-1 is a core component of the IFT particle complex B. (A) IFT70/CrDYF-1 comigrates with other IFT particle subunits at 16S. Flagellar matrix was fractionated through a 12-ml 10–25% sucrose density gradient. The gradient fractions were separated on 10% SDS-PAGE gels and stained with Coomassie Blue. IFT70/CrDYF-1 migrates between IFT72 and IFT57. The lane labeled “pellet” is collected from the bottom of the gradient. (B) IFT70/CrDYF-1 coimmunoprecipitates with other IFT particle complex B proteins. Immunoprecipitates with antibodies against IFT proteins from the flagellar membrane plus matrix were separated on 8% polyacrylamide gels and analyzed by Western blotting. The antibodies used for immunoprecipitation are listed above the Western blots. The antibodies used for Western blotting are indicated on the left. Nonspecific bands are indicated by arrowheads on the left. The band just above IFT81 may come from α-IFT139 antibody, because no such band exists in the starting membrane plus matrix material. The band just below IFT81 apparently comes from protein A beads, as it is present in the beads alone control. (C) Flagellar matrix was treated with or without high salt as described previously (Lucker et al., 2005) and fractionated through a 12-ml 10–25% sucrose density gradient. The sucrose density gradient fractions were separated by 10% SDS-PAGE and analyzed by Western blotting. The arrows mark the peaks of complexes A (left) and B (right).
Figure 4.
Figure 4.
Coexpression and tandem purification of recombinant MBP-IFT70/CrDYF-1 and SIIT-IFT46. Shown here is the Coomassie Blue stained gel of samples from each step of the tandem purification; the first two lanes following the markers contain the insoluble and soluble fractions of bacterial cell lysates. Note that both proteins copurify after tandem purification with amylose and StrepTactin affinity resin. F/T stands for the flow-through proteins that did not bind to the resistive resins.
Figure 5.
Figure 5.
IFT70/CrDYF-1 is partially associated with the axoneme. (A) Procedure for the preparation of the flagellar fractions. This procedure is essentially the same as described in Huang et al. (2007). (B) Stoichiometrically equivalent levels of whole flagella, matrix, membrane plus axoneme, membrane proteins, and bare axoneme (labeled on the top) were isolated according to the procedure described in A. Note that, like other IFT particle proteins, a portion of IFT70/CrDYF-1 remains associated with the axoneme. The samples were separated by 10% SDS-PAGE and analyzed by Western blotting.
Figure 6.
Figure 6.
The IFT70/CrDYF-1 knockdown cells have reduced levels of IFT complex B proteins and shortened flagella. (A) Immunoblots (top panels, WB) of the whole cell extracts isolated from the knockdown cells Ri-6 and Ri-41 and control (wild-type) cells. Letters A and B represent the IFT particle complexes A and B, respectively. The bottom panel is part of a Coomassie Blue stained gel (Gel) to show the equal loading of all the samples. (B) Dual-staining of Ri-6 and control cells with antibodies against α-tubulin (red) and FLA10 (green). Ri-6 cells have much shorter flagella than the control cells. The FLA10 localization pattern in Ri-6 cells appears normal.
Figure 7.
Figure 7.
IFT70/CrDYF-1 is required for flagellar assembly. (A) Levels of cellular IFT particle complex A proteins are elevated while Complex B proteins are reduced when IFT70/CrDYF-1 is reduced. The upper panels show the Western-blotting (WB) results of a few IFT particle proteins of whole cell extracts isolated from the knockdown strains miRNA-1 and miRNA-4, and the control cw92 cells. The IFT70/CrDYF-1 protein is indicated by an arrow on the left. The α-IFT70/CrDYF-1 antibody also recognized a non-specific band indicated by an asterisk (*) on the right. The lower panel is part of a Coomassie Blue-staining gel (Gel) to show the equal loading of all the samples. (B) IFT70/CrDYF-1 reduced miRNA cells assemble short flagella. The plot shows flagellar length distribution of cw92 (n = 116), miRNA-1 (n = 116), and miRNA-4 (n = 108) cells. The mean lengths of the flagella are listed.
Figure 8.
Figure 8.
The flagella of IFT70/CrDYF-1 knockdown cells display a normal ultrastructure. (A) Cross section of a flagellum of a C. reinhardtii cw92 cell showing the 9 + 2 microtubule architecture of the axoneme. (B–G) Electron micrographs of flagella of IFT70/CrDYF-1 knockdown cells (strain miRNA-4). (B–D) Cross sections of flagella. IFT-trains (arrowheads) attached to the B-subfiber of outer-doublets are visible. (E) Longitudinal section of a flagellum with a visible IFT-train (arrowhead). (F) Cross section through a transition zone showing a normal ultrastructure. (G) Cross section through the distal tip of a flagellum. An IFT-train is visible (arrowhead). Scale bar, (A–G), 100 nm.

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