Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 15;28(12):1652-1666.
doi: 10.1091/mbc.E17-01-0017. Epub 2017 Apr 20.

RABL2 Interacts With the Intraflagellar transport-B Complex and CEP19 and Participates in Ciliary Assembly

Affiliations
Free PMC article

RABL2 Interacts With the Intraflagellar transport-B Complex and CEP19 and Participates in Ciliary Assembly

Yuya Nishijima et al. Mol Biol Cell. .
Free PMC article

Abstract

Proteins localized to the basal body and the centrosome play crucial roles in ciliary assembly and function. Although RABL2 and CEP19 are conserved in ciliated organisms and have been implicated in ciliary/flagellar functions, their roles are poorly understood. Here we show that RABL2 interacts with CEP19 and is recruited to the mother centriole and basal body in a CEP19-dependent manner and that CEP19 is recruited to the centriole probably via its binding to the centrosomal protein FGFR1OP. Disruption of the RABL2 gene in Chlamydomonas reinhardtii results in the nonflagellated phenotype, suggesting a crucial role of RABL2 in ciliary/flagellar assembly. We also show that RABL2 interacts, in its GTP-bound state, with the intraflagellar transport (IFT)-B complex via the IFT74-IFT81 heterodimer and that the interaction is disrupted by a mutation found in male infertile mice (Mot mice) with a sperm flagella motility defect. Intriguingly, RABL2 binds to CEP19 and the IFT74-IFT81 heterodimer in a mutually exclusive manner. Furthermore, exogenous expression of the GDP-locked or Mot-type RABL2 mutant in human cells results in mild defects in ciliary assembly. These results indicate that RABL2 localized to the basal body plays crucial roles in ciliary/flagellar assembly via its interaction with the IFT-B complex.

Figures

FIGURE 1:
FIGURE 1:
Interaction between RABL2 and CEP19. (A) Interaction of RABL2 with CEP19. HEK293T cells were transiently cotransfected with expression vectors for EGFP-CEP19 and RABL2B(WT)-HA or its mutant (S35N, Q80L, or D73G). At 24 h after the transfection, lysates were prepared from the transfected cells and immunoprecipitated with GST-fused anti–GFP Nb prebound to glutathione–Sepharose 4B beads. Proteins bound to the precipitated beads were subjected to SDS–PAGE and immunoblotting analysis using anti-HA or anti-GFP antibodies. (B) Schematic representation of the structures of CEP19 and its deletion constructs. (C) RABL2 interacts with the C-terminal region of CEP19. Lysates prepared from HEK293T cells transfected with expression vectors for RABL2B-HA and EGFP, or EGFP-tagged CEP19(WT) or its deletion construct, as indicated, were processed for immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis, as described for A.
FIGURE 2:
FIGURE 2:
Colocalization of RABL2 and CEP19 at the mother centriole and the ciliary base. (A–C) Localization of RABL2 and CEP19 at one of the centrioles. hTERT-RPE1 cells were double stained for RABL2 and γ-tubulin (A–A′′), CEP19 and γ-tubulin (B–B′′), or RABL2 and CEP19(C–C′′). (D–F) Colocalization of RABL2 and CEP19 with ODF2. hTERT-RPE1 cells were double stained for RABL2 and ODF2 (D–D′′), CEP19 and ODF2 (E–E′′), or ODF2 and γ-tubulin (F–F′′). (G–G′′′) Colocalization of RABL2 and CEP19 at the ciliary base. hTERT-RPE1 cells were cultured under serum-starved conditions for 24 h and processed for triple immunostaining for RABL2, CEP19, and Ac-α-tubulin. (H–K) Localization of exogenously expressed RABL2B and its mutants. hTERT-RPE1 cells were transiently cotransfected with expression vectors for tRFP-CEP19 and RABL2B(WT)-EGFP (H–H′′′) or its mutant, S35N (I–I′′′), Q80L (J–J′′′), or D73G (K–K′′′) and stained with an anti–γ-tubulin antibody. (L–O) Localization of CEP19 deletion constructs. hTERT-RPE1 cells were transfected with an expression vector for EGFP-tagged CEP19(WT) (L–L′′) or its deletion construct, as indicated (L–L′′, M–M′′, N–N′′, or O–O′′), and stained with an anti–γ-tubulin antibody. Insets. enlarged images of the boxed regions. Scale bars, 10 µm.
FIGURE 3:
FIGURE 3:
Interaction between RABL2, CEP19, and FGFR1OP. (A) Schematic representation of the structures of FGFR1OP and FOR20. (B) Interaction of CEP19 with FGFR1OP. HEK293T cells were transiently cotransfected with expression vectors for mChe-CEP19 and either EGFP, EGFP-FGFR1OP, or EGFP-FOR20. At 24 h after the transfection, lysates were prepared from the transfected cells and immunoprecipitated with GST–anti-GFP Nb, followed by immunoblotting analysis with antibodies against mRFP or EGFP. (C) FGFR1OP interacts with the CEP19 N-terminal region. Lysates prepared from HEK293T cells transfected with expression vectors for tRFP-FGFR1OP and EGFP, or EGFP-tagged CEP19(WT) or its deletion construct, as indicated, were subjected to immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis with anti-tRFP or anti-GFP antibodies. (D) Tripartite interaction of RABL2, CEP19, and FGFR1OP. hTERT-RPE1 cells were transfected with expression vectors for EGFP-FGFR1OP and either mChe-CEP19 or RABL2-mChe, or both mChe-CEP19 and RABL2-mChe. Lysates from the transfected cells were subjected to immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis with anti-mRFP or anti-GFP antibodies. (E–E′′′) Colocalization of RABL2, CEP19, and FGFR1OP at one of the centrioles. hTERT-RPE1 cells were triple stained for RABL2, CEP19, and FGFR1OP. RABL2 and CEP19 were colocalized to one of the two FGFR1OP-positive centrioles. Insets, enlarged images of the boxed regions. Scale bar, 10 µm.
FIGURE 4:
FIGURE 4:
FGFR1OP interacts with CEP19 and CEP350 via its N- and C-terminal regions, respectively. (A) Schematic representation of the structures of FGFR1OP and its deletion constructs. (B) FGFR1OP interacts with CEP19 via its C-terminal region. Lysates of HEK293T cells transfected with expression vectors for tRFP-CEP19 and EGFP, or EGFP-tagged FGFR1OP(WT) or its deletion construct, as indicated, were subjected to immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis with anti-tRFP or anti-GFP antibodies. (C–G) Localization of FGFR1OP deletion constructs. hTERT-RPE1 cells were transfected with an expression vector for EGFP-tagged FGFR1OP(WT) (C–C”) or its deletion construct (D–D”, E–E”, F–F”, or G–G”) and stained with an anti–γ-tubulin antibody. Insets, enlarged images of the boxed regions. Scale bar, 10 µm. (H) FGFR1OP interacts with CEP350 via its N-terminal region. Lysates from HEK293T cells transfected with an expression vector for mChe-CEP350(3071-3117) together with that for EGFP, EGFP-tagged FGFR1OP(WT), or its deletion construct, as indicated, were subjected to immunoprecipitation with GST–anti-GFP Nb, followed by immunoblotting analysis with anti-mRFP or anti-GFP antibodies.
FIGURE 5:
FIGURE 5:
Loss of centriolar localization of RABL2 in CEP19-KO cells. Control RPE1 cells (A, D, G, J) and the CEP19-KO cell lines 19-1-2 (B, E, H, K) and 19-1-12 (C, F, I, L) were double immunostained for CEP19 (A–C), RABL2 (D–F), or FGFR1OP (G–I) and γ-tubulin (A′–I′) or triple immunostained for IFT88 (J–L) and Ac-α-tubulin plus FGFR1OP (J′–L′). Insets, enlarged images of the boxed regions. Scale bars, 10 µm. (M) Lysates prepared from control RPE1 cells (lane 1) or the CEP19-KO cell line 19-1-2 (lane 2) or 19-1-12 (lane 3) were processed for immunoblotting analysis using antibody against RABL2 (top), IFT88 (middle), or actin (bottom). (N) Cells with centrosomal RABL2 signals in the experiments in D–F were counted, and the percentages of RABL2-positive cells in each condition are shown as a bar graph. Values are means of three independent experiments. In each set of experiments, 30 cells were analyzed, and the total number of analyzed cells (n) is shown. (O) Localization of IFT88 in individual control and CEP19-KO cells was classified as “ciliary base + within cilia,” “mainly ciliary base,” and “no ciliary localization” and counted. The percentages of these populations are expressed as stacked bar graphs. Values are means ± SE of three independent experiments. In each set of experiments, 35–49 ciliated cells were observed, and the total number of ciliated cells observed (n) is shown. **p < 0.0001 (Pearson’s χ2 test).
FIGURE 6:
FIGURE 6:
Decreased ciliogenesis efficiency in CEP19-KO cells. (A–D) Requirement of CEP19 for RABL2 centrosomal localization. CEP19-KO 19-1-2 cells stably expressing EGFP (A–A′′′), EGFP-CEP19(WT) (B–B′′′), EGFP-CEP19(1-120) (C–C′′′), or EGFP-CEP19(91-167) (D–D′′′) were double immunostained for RABL2 (A′–D′) and γ-tubulin (A′′–D′′). Insets, enlarged images of the boxed regions. (E–G) Ciliogenesis in CEP19-KO cells. Control RPE1 cells (E) or the CEP19-KO cell lines 19-1–2 (F) and 19-1-12 (G) were serum starved for 24 h and double immunostained for Ac-α-tubulin (E–G) and γ-tubulin (E′–G′). (H–K) Rescue experiments using CEP19 deletion constructs. CEP19-KO 19-1-12 cells stably expressing EGFP (H–H′′′), EGFP-CEP19(WT) (I–I′′′), EGFP-CEP19(1-120) (J–J′′′), or EGFP-CEP19(91-167) (K–K′′′) were serum starved for 24 h and double immunostained for Ac-α-tubulin (H′–K′) and γ-tubulin (H′′–K′′). Scale bars, 10 µm. (L) Cells with centrosomal RABL2 signals in the experiments shown in A–D were counted, and percentages of RABL2-positive cells are shown as a bar graph. Values are means of three independent experiments. In each set of experiments, 30 cells were analyzed, and the total number of analyzed cells (n) is shown. (M) Ciliated cells in the experiments in E–G were counted, and the percentages of ciliated cells are shown as a bar graph. Values are means ± SE of three independent experiments. In each set of experiments, 38–50 cells were analyzed, and the total number of analyzed cells (n) is shown. **p < 0.001 (Student’s t test). (N) Ciliated cells in the experiments in H–K were counted, and the percentages of ciliated cells are shown as a bar graph. Values are means ± SE of three independent experiments. In each set of experiments, 37–66 cells were analyzed, and the total number of analyzed cells (n) is shown. **p < 0.001; *p < 0.01 (Student’s t test).
FIGURE 7:
FIGURE 7:
Chlamydomonas RABL2 mutant. (A–C) The C. reinhardtii wild-type (WT) strain (A; CC-4533 cw15 mt-) and rabl2 strain (B; LMJ.RY0402.205222) and the rabl2 strain transformed with a RABL2 expression vector (C) were stained with an anti–Ac-α-tubulin antibody and DAPI. (D) Schematic representation of the integration of the paromomycin (Paro) resistance gene cassette in the RABL2 locus (top) and an alignment of the mutant allele sequence determined by direct sequencing of the genomic PCR products with the reference sequence encompassing the coding sequence of exon 6 (bottom). The magenta arrow indicates the direction of Paro cassette integration. (E) RT-PCR analysis of RABL2 mRNA expression in the WT strain (lane 2), the rabl2 strain (lane 3), and the transformant (lane 4). Lane 1 is a DNA size marker (DdeI-digested pSP64 DNA). (F) Flagellated cells in the experiments in A–C were counted, and the percentages of flagellated cells are shown as a bar graph. The number of observed cells (n) is shown in the graph.
FIGURE 8:
FIGURE 8:
Mutually exclusive interactions of RABL2 with the IFT-B complex and CEP19. (A) Interaction of RABL2B(Q80L) with the IFT-B complex demonstrated by the VIP assay. An expression vector for EGFP-IFT22 (as a positive control), EGFP (as a negative control), or an EGFP-tagged RABL2B construct, as indicated, was cotransfected with expression vectors for all IFT-B subunits tagged with mChe or tRFP into HEK293T cells. Cell lysates were immunoprecipitated with GST–anti-GFP Nb prebound to glutathione–Sepharose beads and observed under a microscope as described in Materials and Methods. (B) Interaction of RABL2B(Q80L) with the IFT-B core 1 subunits. Lysates prepared from HEK293T cells coexpressing RABL2B(Q80L)-EGFP and all IFT-B, all core, all core 1, all core 2, all peripheral, or all connecting (IFT38/IFT52/IFT57/IFT88) subunits tagged with mChe or tRFP were processed for the VIP assay as described. (C) Interaction of RABL2B(Q80L) with IFT74 and IFT81 demonstrated by the subtractive VIP assay. Lysates of HEK293T cells coexpressing RABL2B(Q80L)-EGFP and all but one (as indicated) of the IFT-B core 1 subunits tagged with mChe were processed for the VIP assay. (D, E) Interaction of RABL2B(Q80L) with the IFT74–IFT81 dimer. Lysates from HEK293T cells coexpressing RABL2B(Q80L)-EGFP and mChe-IFT74 or mChe-IFT81 alone or both mChe-IFT74 and mChe-IFT81 were processed for VIP assay (D) or immunoblotting analysis with an anti-mRFP or anti-GFP antibody (E). (F, G) Interaction of RABL2 with the IFT74–IFT81 dimer in its GTP-bound state. HEK293T cells coexpressing mChe-IFT74+IFT81 and EGFP-tagged RABL2B(WT), RABL2B(S35N), RABL2B(Q80L), or RABL2B(D73G) were lysed and processed for the VIP assay (F) or immunoblotting analysis with an anti-mRFP or anti-GFP antibody (G). (H–K) Mutually exclusive interactions of RABL2 with the IFT-B complex and CEP19. Lysates prepared from HEK293T cells coexpressing EGFP-IFT74+81 and either RABL2B(Q80L)-mChe or mChe-CEP19 alone, or both RABL2B(Q80L)-mChe and mChe-CEP19 (H, I), or those coexpressing reciprocal combinations of the EGFP and mChe tags (J and K) were processed for the VIP assay (H, J) or immunoblotting analysis with an anti-mRFP or anti-GFP antibody (I, K). (L) Schematic representation of the overall architecture of the IFT-B complex and interactions of RABL2 with CEP19 and the IFT-B complex.
FIGURE 9:
FIGURE 9:
RABL2 participates in ciliary assembly. (A–F) RABL2 does not accumulate at the ciliary tip regardless of impaired retrograde trafficking. Control hTERT-RPE1 cells (A, D) or the IFT139-KO cell line 139-2-6 (B, E) or 139-2–8 (C, F) was cultured under serum-starved conditions to induce ciliogenesis and triple immunostained for RABL2 (A–C), IFT88, and Ac-α-tubulin (A′–C′), or for CEP19 (D–F), Ac-α-tubulin, and γ-tubulin (D′–F′). Insets, enlarged images of the boxed regions. Scale bars, 10 µm. (G–L) Exogenous expression of RABL2 mutants defective in IFT-B binding suppress ciliogenesis. hTERT-RPE1 cells were transfected with an expression vector for EGFP (G) or EGFP-tagged RABL2B(WT) (H), RABL2B(S35N) (I), RABL2B(Q80L) (J), or RABL2B(D73G) (K) and cultured for 12 h under normal conditions. The cells were then cultured under serum-starved conditions for 12 h and processed for immunostaining with antibodies against Ac-α-tubulin and γ-tubulin (G′–K′). (L) Ciliated cells in the experiments in G–K were counted, and percentages of ciliated cells are expressed as a bar graph. Values are means ± SE of three independent experiments. In each set of experiments, 30–33 cells were analyzed, and the total number of cells analyzed for each condition (n) is shown. **p < 0.005; *p < 0.02 (Student’s t test).

Similar articles

See all similar articles

Cited by 16 articles

See all "Cited by" articles

References

    1. Ahmed NT, Gao C, Lucker BF, Cole DG, Mitchell DR. ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. J Cell Biol. 2008;183:313–322. - PMC - PubMed
    1. Bhogaraju S, Cajánek L, Fort C, Blisnick T, Weber K, Taschner M, Mizuno N, Lamla S, Bastin P, Nigg EA, Lorentzen E. Molecular basis of tubulin transport within the cilium by IFT74 and IFT81. Science. 2013;341:1009–1012. - PMC - PubMed
    1. Bhogaraju S, Weber K, Engel BD, Lechtreck K-F, Lorentzen E. Getting tubulin to the tip of the cilium: one IFT train, many different tubulin cargo binding sites? Bioessays. 2014;36:463–467. - PubMed
    1. Brown JM, Witman GB. Cilia and diseases. BioScience. 2014;64:1126–1137. - PMC - PubMed
    1. Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL. Chlamydomonas kinesin-II–dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol. 1998;141:993–1008. - PMC - PubMed

MeSH terms

LinkOut - more resources

Feedback