Rab35 controls cilium length, function and membrane composition
2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.15252/embr.201847625
ISSN1469-3178
AutoresStefanie Kuhns, Cecília Seixas, Sara Pestana, Bárbara Tavares, Renata Nogueira, Raquel Jacinto, José S. Ramalho, Jeremy C. Simpson, Jens Andersen, Arnaud Échard, Susana S. Lopes, Duarte C. Barral, Oliver E. Blacque,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle21 August 2019Open Access Source DataTransparent process Rab35 controls cilium length, function and membrane composition Stefanie Kuhns Corresponding Author Stefanie Kuhns [email protected] orcid.org/0000-0002-3065-7818 School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark Search for more papers by this author Cecília Seixas Corresponding Author Cecília Seixas [email protected] orcid.org/0000-0002-7364-4320 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Sara Pestana Sara Pestana orcid.org/0000-0002-0995-0040 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Bárbara Tavares Bárbara Tavares CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Renata Nogueira Renata Nogueira CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Raquel Jacinto Raquel Jacinto orcid.org/0000-0002-4029-0204 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author José S Ramalho José S Ramalho CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Jeremy C Simpson Jeremy C Simpson School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland Search for more papers by this author Jens S Andersen Jens S Andersen orcid.org/0000-0002-6091-140X Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark Search for more papers by this author Arnaud Echard Arnaud Echard orcid.org/0000-0001-7402-1398 Institut Pasteur and CNRS UMR3691, Paris, France Search for more papers by this author Susana S Lopes Susana S Lopes CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Duarte C Barral Corresponding Author Duarte C Barral [email protected] orcid.org/0000-0001-8867-2407 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Oliver E Blacque Corresponding Author Oliver E Blacque [email protected] orcid.org/0000-0003-1598-2695 School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland Search for more papers by this author Stefanie Kuhns Corresponding Author Stefanie Kuhns [email protected] orcid.org/0000-0002-3065-7818 School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark Search for more papers by this author Cecília Seixas Corresponding Author Cecília Seixas [email protected] orcid.org/0000-0002-7364-4320 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Sara Pestana Sara Pestana orcid.org/0000-0002-0995-0040 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Bárbara Tavares Bárbara Tavares CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Renata Nogueira Renata Nogueira CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Raquel Jacinto Raquel Jacinto orcid.org/0000-0002-4029-0204 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author José S Ramalho José S Ramalho CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Jeremy C Simpson Jeremy C Simpson School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland Search for more papers by this author Jens S Andersen Jens S Andersen orcid.org/0000-0002-6091-140X Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark Search for more papers by this author Arnaud Echard Arnaud Echard orcid.org/0000-0001-7402-1398 Institut Pasteur and CNRS UMR3691, Paris, France Search for more papers by this author Susana S Lopes Susana S Lopes CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Duarte C Barral Corresponding Author Duarte C Barral [email protected] orcid.org/0000-0001-8867-2407 CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Oliver E Blacque Corresponding Author Oliver E Blacque [email protected] orcid.org/0000-0003-1598-2695 School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland Search for more papers by this author Author Information Stefanie Kuhns *,1,2,‡, Cecília Seixas *,3,‡, Sara Pestana3, Bárbara Tavares3, Renata Nogueira3, Raquel Jacinto3, José S Ramalho3, Jeremy C Simpson4, Jens S Andersen2, Arnaud Echard5, Susana S Lopes3, Duarte C Barral *,3,‡ and Oliver E Blacque *,1,‡ 1School of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark 3CEDOC, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal 4School of Biology and Environmental Science, University College Dublin, Dublin 4, Ireland 5Institut Pasteur and CNRS UMR3691, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +45 65 50 71 86; E-mail: [email protected] *Corresponding author. Tel: +351 218 803 101; E-mail: [email protected] *Corresponding author. Tel: +351 218 803 101; E-mail: [email protected] *Corresponding author. Tel: +353 1 7166953; E-mail: [email protected] EMBO Reports (2019)20:e47625https://doi.org/10.15252/embr.201847625 [The copyright line of this article was changed on 25 January 2021 after original online publication.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators. Synopsis Rab35 at the mammalian and zebrafish cilium regulates cilium length and left-right asymmetry, and controls the ciliary levels of PI(4,5)P2 and regulators of the Sonic hedgehog pathway. Rab35 GTPase localizes to the ciliary membrane and pocket of mammalian cells and Kupffer's vesicles of zebrafish along with its GAP and GEF. Rab35 depletion leads to formation of shorter cilia in both systems and interferes with left- right symmetry formation of zebrafish. Rab35 depletion reduces the levels of Sonic hedgehog signaling components and PI(4,5)P2 in the ciliary membrane. Rab35 interacts with Arl13b and inhibits its ciliary accumulation. Introduction Primary cilia are microtubule-based organelles that protrude from the surface of most vertebrate cell types. Operating as antenna-like structures, primary cilia detect and transmit chemical, light and mechanical signals from the extracellular environment to the intracellular space 1. Primary cilia are also critical for embryonic and postnatal development, serving key roles in important cell–cell communication signalling pathways (e.g. Sonic hedgehog, Wnt, PDGFα) 2. For example, in the limb bud and developing nervous system, bone and neural tube patterning relies on the trafficking of Sonic hedgehog (Shh) signalling proteins into and out of cilia 3. At the left-right organiser (LRO), or the equivalent Kupffer's vesicle in zebrafish, the correct ratio of immotile and motile cilia 4 direct left-right patterning of the body plane via mechanisms that involve directional fluid flow, mechano- or chemo-sensation and planar cell polarity (PCP) signalling 5-7. Not surprisingly, defects in primary and motile cilia cause a wide range of mono- or multi-symptomatic "ciliopathy" disorders such as primary ciliary dyskinesia, situs inversus, polycystic kidney disease, Joubert syndrome (JBTS), Meckel–Gruber syndrome (MKS) and Bardet–Biedl syndrome (BBS), which, collectively, affect most body tissues 8, 9. Regulation of cilium structure, function and molecular composition is heavily dependent on various intracellular transport pathways. Chief amongst these is intraflagellar transport (IFT), which operates bidirectionally along the ciliary microtubules and is driven by kinesin-2 and IFT dynein motors, together with IFT-A, IFT-B and BBSome cargo adaptor complexes 10-13. Cilium structure and organisation are also reliant on cytosolic and membrane diffusion barriers at the ciliary base, as well as on secretory, exocytic (e.g. ciliary ectosome release), endocytic and recycling pathways that control ciliary membrane homeostasis 14-24. Together, these transport and barrier-associated processes dynamically control the molecular composition of the ciliary membrane and cytosol and, therefore, the appropriate sensory and signalling output of the organelle. Intracellular membrane trafficking is highly regulated by members of the Rab and Arf [including Arl (Arf-like)] families of guanine nucleotide-binding proteins (G proteins) 25-28. Acting as molecular switches, alternating between inactive GDP- and active (effector binding) GTP-bound states, G proteins regulate vesicular membrane traffic steps to ensure correct cargo transport via integrated sorting, delivery, uptake and recycling pathways. G protein activity is tightly regulated, with guanine nucleotide exchange factors (GEFs) promoting GTP binding, and GTPase-activating proteins (GAPs) enhancing the G protein's intrinsic GTPase activity which, when present, hydrolyses GTP to GDP 29. Currently, at least nine of the 66 Rabs that comprise this family of GTPases (Rab 5/8/10/11/17/23/28/29/34), as well as 3 Rab-like proteins (Rabl2/4/5), are linked to cilium formation and/or function, and the control of ciliary membrane protein levels 30-34. For example, vertebrate and mammalian Rab11-Rabin8-Rab8 cascades are involved in early steps of cilium formation, and rhodopsin transport to the photoreceptor cell cilium (outer segment) 35-37. Furthermore, a number of cilium-localised Rab, Rab-like and Arl proteins are associated with IFT and the BBSome, and the trafficking of Hedgehog signalling intermediates out of cilia 32, 38-40. Amongst Arf/Arl proteins, ciliary membrane-restricted Arl13b is linked to ciliogenesis, Shh signal regulation, IFT regulation, and the localisation and distribution of multiple ciliary proteins such as the inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase, INPP5E, in part via its role as a GEF of Arl3 41-52. Consistent with their important cilia-related functions, a number of Arls and Rabs such as ARL13B, ARL3, ARL6, RAB23 and RAB28 are mutated in ciliopathy disorders 53-57. With the aim of investigating new Rab proteins that regulate cilium formation and/or function, we focused on Rab35, a plasma membrane and endosomal protein with roles in cargo recycling, cytokinesis, actin cytoskeleton regulation, and autophagy, amongst others 58-68. Initial clues towards a Rab35 ciliary role include its presence in a photoreceptor outer segment proteome, and high-throughput siRNA or CRISPR screens implicating Rab35 as either a positive or negative regulator of ciliogenesis, or a regulator of Hedgehog signalling in NIH3T3 mouse fibroblasts 33, 69-72. Here, using mammalian cell culture and zebrafish models, we show that Rab35 localises to the ciliary membrane and regulates primary and LRO cilium length. We also reveal that Rab35 controls the ciliary levels of Shh signal regulators, Smoothened, Arl13b, and INPP5E, as well as the INPP5E target, PI(4,5)P2. Furthermore, we identify the Rab35 GEF and GAP, DENND1B and TBC1D10A, respectively, as the regulators of Rab35 in the ciliary context. Together, our data uncover a novel conserved role for Rab35 in controlling cilium length and the ciliary levels of mammalian Shh signalling regulators. Results Rab35 localises to the ciliary membrane Rab35 has been found in several proteomes of mammalian primary cilia 70, 71, 73. To more directly investigate a possible ciliary localisation of Rab35, we employed two well-established mammalian cell models, namely mouse renal epithelial (IMCD3) and human retinal pigment epithelial (hTERT-RPE1) cells, where ciliogenesis can be induced by serum withdrawal 74, 75. First, we analysed mouse IMCD3 and human hTERT-RPE1 cells transiently expressing GFP-tagged Rab35 and found that it localises along the ciliary axoneme in ~60% of transfected ciliated cells (Fig 1A and B). We also observed GFP-Rab35 at the plasma membrane and in vesicular structures, consistent with previously described localisations in other cell types and its known endosomal functions 58, 60-64, 66. To further analyse the ciliary association of Rab35, we established a stable hTERT-RPE1 cell line expressing GFP-RAB35 and performed super-resolution microscopy to localise RAB35 in relation to markers of the ciliary membrane (ARL13B) and axoneme (IFT88 and acetylated tubulin; Fig 1C). The GFP-RAB35 signal coincides with that of ARL13B, with the radial extent of both signals being wider than that of the axonemal markers, indicating association of RAB35 with the ciliary membrane. Notably, in the proximal-most part of the cilium, the radial extent of the GFP-RAB35 signal is wider than that of ARL13B (Figs 1C and D, and EV1A and B), and in ~20% of cells, GFP-RAB35 is also more concentrated in this region (Fig EV1C). This proximal staining is reminiscent of the ciliary pocket localisation for the membrane remodelling Eps15 homology domain (EHD) 1 protein 76 and super-resolution imaging of GFP-RAB35 expressing hTERT-RPE1 cells stained for endogenous EHD1 revealed co-localisation of both proteins in the proximal region of the cilium; importantly, these EHD1/GFP-RAB35 co-localising signals appeared broader than the radial extent of the ARL13B ciliary membrane signal, indicative of their association with the ciliary pocket membrane (Fig 1E). Taken together, these data suggest that GFP-RAB35 localises to the ciliary pocket in addition to the ciliary membrane. Figure 1. Rab35 localises to the ciliary membrane Localisation of transiently expressed GFP-Rab35 in IMCD3 cells after 48 h serum starvation and staining for acetylated tubulin (acetyl. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 μm. Localisation of transiently expressed GFP-RAB35 in hTERT-RPE1 cells after 24 h serum starvation and staining for polyglutamylated tubulin (polyglu. tub.) and DNA. Insets show higher magnification images of the cilia region. Scale bars, 10 μm. Super-resolution (FV-OSR) imaging of hTERT-RPE1 stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP, ARL13B (cilia membrane), IFT88 (axoneme), acetylated tubulin (acetyl. tub.; axoneme), and γ-tubulin (γ-tub; centrosome). Top panels show representative images, and bottom panel shows line profile plots of fluorescence intensity (arbitrary units; a. u.) in the distal (dotted line) and proximal (dashed line) cilia regions. Data are mean ± SEM (n = 5 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating SEM values. Scale bars, 1 μm. Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, ARL13B and acetylated tubulin. Right panels show a cilium with proximal enrichment of GFP-RAB35 and left panels a cilium with even GFP-RAB35 localisation along the full length. Scale bars, 1 μm. Representative FV-OSR images of 24 h serum-starved hTERT-RPE1 stably expressing GFP-RAB35 and stained for GFP, EHD1 and ARL13B. Line profile plots of fluorescence intensity in the proximal cilia region (dashed line) are shown to the right. Data are mean ± SEM (n = 6 cilia), with the solid line in the line profile plots indicating mean and the dotted lines indicating SEM values. Scale bars, 1 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Rab35 localises to the ciliary membrane A, B. Super-resolution (FV-OSR) imaging of hTERT-RPE1 stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP and acetylated tubulin (acetyl. tub.) (A) or GFP, ARL13B and polyglutamylated tubulin (polyglu. tub.). Scale bars, 1 μm. C. Representative images of hTERT-RPE1 cells stably expressing GFP-RAB35 after 24 h serum starvation and staining for GFP, ARL13B, polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown to the bottom. Scale bars, 10 μm. Graph to the right shows quantification of cilia with GFP-RAB35 localisation to the proximal region of the cilium marked with polyglutamylated tubulin staining (ARL13B > GFP-RAB35 = polyglu. tub.) and cilia with GFP-RAB35 localisation along the full length of the cilium marked with ARL13B staining (ARL13B = GFP-RAB35 > polyglu. tub.). Note that in ˜15% of cilia all three markers stained the same cilia region (ARL13B = GFP-RAB35 = polyglu. tub.). Data are mean ± SEM of three independent experiments (n ≥ 30 cilia per experimental condition). Download figure Download PowerPoint Rab35 regulates cilium length The ciliary localisation of Rab35 suggests that this G protein may regulate cilium formation, structure or function. Indeed, there are recent suggestions of a role for Rab35 in Hedgehog signalling regulation 33, 72; also, Rab35 was identified in an siRNA screen as a positive ciliogenesis modulator 69, although additional high-throughput siRNA screening studies indicate that Rab35 is either a negative regulator of cilium formation 71, or plays no role at all in this process 77. To shed light on these seemingly contradictory results and test for a potential role of Rab35 in ciliogenesis, we employed siRNA-mediated depletion of RAB35 in hTERT-RPE1 cells and analysed cilia using acetylated tubulin staining. Two independent siRNAs targeting human RAB35 were used, both of which reduce RAB35 expression by > 90% (Fig 2A, and Appendix Fig S1A). Although RAB35 depletion does not affect the number of ciliated hTERT-RPE1 cells observed after serum withdrawal (Fig 2B), the cilia are significantly shorter, with a median cilia length of ~2.1 μm in RAB35-depleted cells, compared to ~2.8 μm in non-depleted cells (Fig 2C and D). Expression of an siRNA-resistant GFP-RAB35 construct rescues the cilia length defect, thus ruling out the possibility that this phenotype is due to off-target effects (Fig 2C and D). Similar results were observed in IMCD3 cells using a pool of siRNAs targeting mouse Rab35, where cilia length is reduced by ~20% (Figs 2E–H, and EV2B and C, and Appendix Fig S1B). To further validate these results and to address the possibility that residual Rab35 due to incomplete siRNA-mediated depletion may still function in ciliogenesis, we generated Rab35 knockout (KO) NIH3T3 cell lines using the CRISPR/Cas9 system 78. We obtained two independent Rab35 KO clones (KO#1 and KO#2) using two different sgRNAs targeting exon 3 and confirmed Rab35 protein loss by immunoblot analysis and genomic locus disruption by sequencing (Fig 2I and Appendix Fig S1C). Analysis of cilia using acetylated tubulin staining revealed no differences in the number of ciliated cells between wild-type (WT) and Rab35 KO NIH3T3 cells; however, like Rab35-depleted cells, the KO cells display a shorter cilium, with a median length of ~1.8 μm compared to ~2.4 μm in the WT control (Fig 2J–L). Collectively, these findings suggest that Rab35 is not involved in initial cilium formation and demonstrate that Rab35 regulates the length of primary cilia. Figure 2. Rab35 regulates cilium length A. Immunoblot analysis of RAB35 depletion in hTERT-RPE1 cells transfected with two independent siRNAs targeting RAB35 (RAB35-1, RAB35-2) or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. β-tubulin served as a loading control. B. Quantification of ciliation of hTERT-RPE1 cells treated as (A) and using acetylated tubulin staining as a cilia marker. Data are mean ± SEM of four independent experiments (n ≥ 50 cilia per experimental condition). C, D. hTERT-RPE1 cells and hTERT-RPE1 cells stably expressing siRNA-resistant GFP-RAB35 were treated as in (A) and stained for GFP, acetylated tubulin (acetyl. tub.) and DNA. Representative images in (C) of cells treated with Neg or RAB35 siRNA. Regions within white boxes shown at higher magnifications to the right. Scale bars, 10 μm. Cilia length quantifications in (D) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal–Wallis followed by Dunn's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.: non-significant; P-values: hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-Neg vs. hTERT-RPE1-RAB35-1 P < 0.0001, hTERT-RPE1-RAB35-1 vs. GFP-RAB35-RAB35-1 P = 0.0249, hTERT-RPE1-RAB35-2 vs. GFP-RAB35-RAB35-2 P = 0.0056). E. Immunoblot analysis of Rab35 depletion in IMCD3 cells transfected with pool of siRNAs targeting mouse Rab35 or a non-targeting siRNA control (Neg). 24 h after transfection, cells were serum-starved for further 48 h. GAPDH served as a loading control. F. Quantification of ciliation of IMCD3 cells treated as in (E) and using acetylated tubulin staining as a cilia marker. Data are mean ± SEM of three independent experiments (n ≥ 50 cilia per experimental condition). G, H. Immunofluorescence of IMCD3 representative images treated as in (E), cells are stained for acetylated tubulin (acetyl. tub.) and DNA. Cilia length quantification in (H) is shown as box-and-whisker plots and is the result of three independent experiments (n ≥ 50 cilia per experimental condition). Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. Statistical significance according to unpaired t-test with Mann–Whitney test (***P = 0.0002). I. Immunoblot analysis of NIH3T3 wild-type (WT) and Rab35 knockout (KO) cell lines. β-tubulin served as a loading control. J–L. NIH3T3 WT and Rab35 KO cell lines were serum-starved for 24 h and stained for acetylated tubulin (acetyl. tub.) and DNA. Quantification of ciliation in (J). Data are mean ± SEM of three independent experiments (n ≥ 100 cilia per experimental condition). Representative images in (K). Regions within white boxes shown at higher magnifications at the bottom. Scale bars, 10 μm. Cilia length quantifications in (L) are shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment of three is shown (n ≥ 100 cilia per experimental condition). Statistical significance according to Kruskal–Wallis followed by Dunn's post hoc test (****P < 0.0001). Source data are available online for this figure. Source Data for Figure 2 [embr201847625-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Ciliary length is regulated by Rab35 nucleotide-bound state A. Representative images of IMCD3 cells transiently expressing wild-type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged Rab35. 12 h after transfection, cells were serum-starved for 48 h and stained for GFP and acetylated tubulin (acetyl. tub.) Higher magnification images of the cilia region shown in smaller panels. Scale bars, 10 μm. B–E. Quantification of ciliary length in μm (B), percentage of ciliation (C) and GFP-Rab35 ciliary localisation (E) in IMCD3 cells transiently expressing the indicated GFP-Rab35 constructs. Cilia length quantification in (B) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. (D) Histogram of cilia length distribution in which three categories of cilia length were considered: [0–1.5 μm length]; [1.5–4 μm], and [4–9 μm]; one representative experiment out of three is shown; n ≥ 50 cilia per experimental condition. Data in (C, E) are mean ± SEM of three independent experiments; n ≥ 100 cilia per experimental condition. Statistical significance according to Kruskal–Wallis followed by Dunn's post hoc test (*P < 0.05, ***P < 0.001; Rab35-WT vs. Rab35-SN P < 0.0001, Rab35-WT vs. Rab35-QL P = 0.0105). Download figure Download PowerPoint Rab35 ciliary localisation and function depend on its nucleotide-bound state Rab GTPases are regulated by their nucleotide-bound state. To assess the requirements of GDP and GTP binding for the ciliary localisation of Rab35, we overexpressed GFP-tagged dominant-negative (GDP-bound) or constitutively active (GTP-bound) mutants in serum-starved hTERT-RPE1 cells 58. Both WT and GTP-bound RAB35-Q67L localise to cilia in ~60% of transfected ciliated cells. In contrast, ciliary signals for GDP-bound RAB35-S22N are much less frequent (~13%; Fig 3A and B). We also assessed whether overexpression of the Rab35 mutants affects hTERT-RPE1 cilium formation and/or structure. We found that RAB35-S22N overexpression exerts a dominant-negative effect on ciliogenesis, resulting in a severe reduction in ciliation (down to 25% of cells; Fig 3C), with those cilia that form being significantly shorter (~20% reduction compared to GFP control; Fig 3D). Conversely, cells overexpressing RAB35-Q67L display an increase in cilium length (Fig 3A and D). We obtained similar results in IMCD3 cells, where ~48% of cells overexpressing GFP-tagged Rab35-S22N exhibit cilia shorter than 1.5 μm (compared with 10 and 20% for cells expressing GFP-tagged Rab35-Q67L or Rab35-WT, respectively), and 33% not forming a cilium (compared with 14% for cells expressing Rab35 WT; Fig EV2A–D). Moreover, like hTERT-RPE1 cells, ciliary localisations were detected more frequently (60% of cilia) for GFP-tagged Rab35-WT and Rab35-Q67L compared to Rab35-S22N (~40% of cilia; Fig EV2E). We conclude from these data that GTP binding to Rab35 is required for its ciliary localisation and function in cilium length regulation. Figure 3. Rab35 ciliary localisation and function depends on its nucleotide-bound state A. Representative images of hTERT-RPE1 cells transiently expressing wild-type (WT), GDP-bound (S22N) or GTP-bound (Q67L) GFP-tagged RAB35. Cells were serum-starved for 24 h and stained for polyglutamylated tubulin (polyglu. tub.) and DNA. Higher magnification images of the cilia region shown in smaller panels. Scale bars, 10 μm. B–D. Quantification of GFP-RAB35 ciliary localisation (B), ciliation (C), and ciliary length in hTERT-RPE1 cells transiently expressing indicated GFP-RAB35 constructs or GFP. Data in (B, C) are mean ± SEM of three independent experiments. Statistical significance according to ANOVA followed by Bonferroni post hoc test (**P < 0.01; P-values: (B) P = 0.0015, (C) P = 0.0062). Cilia length quantification in (D) is shown as box-and-whisker plots. Horizontal lines show 25, 50 and 75th percentiles; whiskers extend to minimum and maximum values. One representative experiment out of three is shown (n ≥ 50 cilia per experimental condition). Statistical significance according to Kruskal–Wallis followed by Dunn's post hoc test (*P < 0.05, **P < 0.01; P-values: GFP vs. GFP-RAB35-S22N P = 0.0015, GFP vs. GFP-RAB35-Q67L P = 0.0158). E, F. Analysis of cilia length in hTERT-RPE1 cells depleted of GEF (DENND1A, DENND1B) an
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