Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

Laboratory of Adaptive Immunity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Forsythe E., and Beales P. L. (2013) Bardet-Biedl syndrome. Eur. J. Hum. Genet. 21, 8–13 10.1038/ejhg.2012.115 PubMed DOI PMC

Niederlova V., Modrak M., Tsyklauri O., Huranova M., and Stepanek O. (2019) Meta-analysis of genotype-phenotype associations in Bardet-Biedl syndrome uncovers differences among causative genes. Hum. Mutat. 40, 2068–2087 10.1002/humu.23862 PubMed DOI

Nachury M. V., Loktev A. V., Zhang Q., Westlake C. J., Peränen J., Merdes A., Slusarski D. C., Scheller R. H., Bazan J. F., Sheffield V. C., and Jackson P. K. (2007) A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129, 1201–1213 10.1016/j.cell.2007.03.053 PubMed DOI

Loktev A. V., Zhang Q., Beck J. S., Searby C. C., Scheetz T. E., Bazan J. F., Slusarski D. C., Sheffield V. C., Jackson P. K., and Nachury M. V. (2008) A BBSome subunit links ciliogenesis, microtubule stability, and acetylation. Dev. Cell 15, 854–865 10.1016/j.devcel.2008.11.001 PubMed DOI

Wingfield J. L., Lechtreck K. F., and Lorentzen E. (2018) Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery. Essays Biochem. 62, 753–763 10.1042/EBC20180030 PubMed DOI PMC

Dilan T. L., Singh R. K., Saravanan T., Moye A., Goldberg A. F. X., Stoilov P., and Ramamurthy V. (2018) Bardet-Biedl syndrome-8 (BBS8) protein is crucial for the development of outer segments in photoreceptor neurons. Hum. Mol. Genet. 27, 283–294 10.1093/hmg/ddx399 PubMed DOI PMC

Nachury M. V. (2018) The molecular machines that traffic signaling receptors into and out of cilia. Curr. Opin. Cell Biol. 51, 124–131 10.1016/j.ceb.2018.03.004 PubMed DOI PMC

Liu P. W., and Lechtreck K. F. (2018) The Bardet-Biedl syndrome protein complex is an adapter expanding the cargo range of intraflagellar transport trains for ciliary export. Proc. Natl. Acad. Sci. U. S. A. 115, E934–E943 10.1073/pnas.1713226115 PubMed DOI PMC

Jin H., White S. R., Shida T., Schulz S., Aguiar M., Gygi S. P., Bazan J. F., and Nachury M. V. (2010) The conserved Bardet-Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia. Cell 141, 1208–1219 10.1016/j.cell.2010.05.015 PubMed DOI PMC

Zhang Q., Seo S., Bugge K., Stone E. M., and Sheffield V. C. (2012) BBS proteins interact genetically with the IFT pathway to influence SHH-related phenotypes. Hum. Mol. Genet. 21, 1945–1953 10.1093/hmg/dds004 PubMed DOI PMC

Guo D. F., Cui H., Zhang Q., Morgan D. A., Thedens D. R., Nishimura D., Grobe J. L., Sheffield V. C., and Rahmouni K. (2016) The BBSome controls energy homeostasis by mediating the transport of the leptin receptor to the plasma membrane. PLoS Genet. 12, e1005890 10.1371/journal.pgen.1005890 PubMed DOI PMC

Nishimura D. Y., Fath M., Mullins R. F., Searby C., Andrews M., Davis R., Andorf J. L., Mykytyn K., Swiderski R. E., Yang B., Carmi R., Stone E. M., and Sheffield V. C. (2004) Bbs2-null mice have neurosensory deficits, a defect in social dominance, and retinopathy associated with mislocalization of rhodopsin. Proc. Natl. Acad. Sci. U. S. A. 101, 16588–16593 10.1073/pnas.0405496101 PubMed DOI PMC

McIntyre J. C., Hege M. M., and Berbari N. F. (2016) Trafficking of ciliary G protein-coupled receptors. Methods Cell Biol. 132, 35–54 10.1016/bs.mcb.2015.11.009 PubMed DOI

Loktev A. V., and Jackson P. K. (2013) Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia. Cell Rep. 5, 1316–1329 10.1016/j.celrep.2013.11.011 PubMed DOI

Berbari N. F., Lewis J. S., Bishop G. A., Askwith C. C., and Mykytyn K. (2008) Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc. Natl. Acad. Sci. U. S. A. 105, 4242–4246 10.1073/pnas.0711027105 PubMed DOI PMC

Woodsmith J., Apelt L., Casado-Medrano V., Özkan Z., Timmermann B., and Stelzl U. (2017) Protein interaction perturbation profiling at amino-acid resolution. Nat. Methods 14, 1213–1221 10.1038/nmeth.4464 PubMed DOI

Katoh Y., Nozaki S., Hartanto D., Miyano R., and Nakayama K. (2015) Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci. 128, 2351–2362 10.1242/jcs.168740 PubMed DOI

Klink B. U., Zent E., Juneja P., Kuhlee A., Raunser S., and Wittinghofer A. (2017) A recombinant BBSome core complex and how it interacts with ciliary cargo. Elife 6, e27434 10.7554/eLife.27434 PubMed DOI PMC

Mourão A., Nager A. R., Nachury M. V., and Lorentzen E. (2014) Structural basis for membrane targeting of the BBSome by ARL6. Nat. Struct. Mol. Biol. 21, 1035–1041 10.1038/nsmb.2920 PubMed DOI PMC

Knockenhauer K. E., and Schwartz T. U. (2015) Structural characterization of Bardet-Biedl syndrome 9 protein (BBS9). J. Biol. Chem. 290, 19569–19583 10.1074/jbc.M115.649202 PubMed DOI PMC

Zhang Q., Yu D., Seo S., Stone E. M., and Sheffield V. C. (2012) Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome. J. Biol. Chem. 287, 20625–20635 10.1074/jbc.M112.341487 PubMed DOI PMC

Klink B. U., Gatsogiannis C., Hofnagel O., Wittinghofer A., and Raunser S. (2020) Structure of the human BBSome core complex. Elife 9, e53910 10.7554/eLife.53910 PubMed DOI PMC

Chou H. T., Apelt L., Farrell D. P., White S. R., Woodsmith J., Svetlov V., Goldstein J. S., Nager A. R., Li Z., Muller J., Dollfus H., Nudler E., Stelzl U., DiMaio F., Nachury M. V., et al. (2019) The molecular architecture of native BBSome obtained by an integrated structural approach. Structure 27, 1384–1394.e4 10.1016/j.str.2019.06.006 PubMed DOI PMC

Singh S. K., Gui M., Koh F., Yip M. C., and Brown A. (2020) Structure and activation mechanism of the BBSome membrane protein trafficking complex. Elife 9, e53322 10.7554/eLife.53322 PubMed DOI PMC

Huranová M., Ivani I., Benda A., Poser I., Brody Y., Hof M., Shav-Tal Y., Neugebauer K. M., and Stanek D. (2010) The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J. Cell Biol. 191, 75–86 10.1083/jcb.201004030 PubMed DOI PMC

Huranova M., Muruganandam G., Weiss M., and Spang A. (2016) Dynamic assembly of the exomer secretory vesicle cargo adaptor subunits. EMBO Rep. 17, 202–219 10.15252/embr.201540795 PubMed DOI PMC

Kremers G. J., Goedhart J., van Munster E. B., and Gadella T. W. Jr. (2006) Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry 45, 6570–6580 10.1021/bi0516273 PubMed DOI

Kim J. C., Badano J. L., Sibold S., Esmail M. A., Hill J., Hoskins B. E., Leitch C. C., Venner K., Ansley S. J., Ross A. J., Leroux M. R., Katsanis N., and Beales P. L. (2004) The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression. Nat. Genet. 36, 462–470 10.1038/ng1352 PubMed DOI

Chamling X., Seo S., Searby C. C., Kim G., Slusarski D. C., and Sheffield V. C. (2014) The centriolar satellite protein AZI1 interacts with BBS4 and regulates ciliary trafficking of the BBSome. PLoS Genet. 10, e1004083 10.1371/journal.pgen.1004083 PubMed DOI PMC

Hernandez-Hernandez V., Pravincumar P., Diaz-Font A., May-Simera H., Jenkins D., Knight M., and Beales P. L. (2013) Bardet-Biedl syndrome proteins control the cilia length through regulation of actin polymerization. Hum. Mol. Genet. 22, 3858–3868 10.1093/hmg/ddt241 PubMed DOI PMC

Gambarotto D., Zwettler F. U., Le Guennec M., Schmidt-Cernohorska M., Fortun D., Borgers S., Heine J., Schloetel J. G., Reuss M., Unser M., Boyden E. S., Sauer M., Hamel V., and Guichard P. (2019) Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat. Methods 16, 71–74 10.1038/s41592-018-0238-1 PubMed DOI PMC

Zhang Q., Nishimura D., Vogel T., Shao J., Swiderski R., Yin T., Searby C., Carter C. S., Kim G., Bugge K., Stone E. M., and Sheffield V. C. (2013) BBS7 is required for BBSome formation and its absence in mice results in Bardet-Biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking. J. Cell Sci. 126, 2372–2380 10.1242/jcs.111740 PubMed DOI PMC

Quarantotti V., Chen J. X., Tischer J., Gonzalez Tejedo C., Papachristou E. K., D'Santos C. S., Kilmartin J. V., Miller M. L., and Gergely F. (2019) Centriolar satellites are acentriolar assemblies of centrosomal proteins. EMBO J. 38, e101082 10.15252/embj.2018101082 PubMed DOI PMC

Dammermann A., and Merdes A. (2002) Assembly of centrosomal proteins and microtubule organization depends on PCM-1. J. Cell Biol. 159, 255–266 10.1083/jcb.200204023 PubMed DOI PMC

Stowe T. R., Wilkinson C. J., Iqbal A., and Stearns T. (2012) The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium. Mol. Biol. Cell 23, 3322–3335 10.1091/mbc.E12-02-0134 PubMed DOI PMC

Seo S., Zhang Q., Bugge K., Breslow D. K., Searby C. C., Nachury M. V., and Sheffield V. C. (2011) A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened. PLoS Genet. 7, e1002358 10.1371/journal.pgen.1002358 PubMed DOI PMC

Marion V., Stutzmann F., Gérard M., De Melo C., Schaefer E., Claussmann A., Hellé S., Delague V., Souied E., Barrey C., Verloes A., Stoetzel C., and Dollfus H. (2012) Exome sequencing identifies mutations in LZTFL1, a BBSome and smoothened trafficking regulator, in a family with Bardet–Biedl syndrome with situs inversus and insertional polydactyly. J. Med. Genet. 49, 317–321 10.1136/jmedgenet-2012-100737 PubMed DOI

Jiang J., Promchan K., Jiang H., Awasthi P., Marshall H., Harned A., and Natarajan V. (2016) Depletion of BBS protein LZTFL1 affects growth and causes retinal degeneration in mice. J. Genet. Genomics 43, 381–391 10.1016/j.jgg.2015.11.006 PubMed DOI PMC

Schmidt T. I., Kleylein-Sohn J., Westendorf J., Le Clech M., Lavoie S. B., Stierhof Y. D., and Nigg E. A. (2009) Control of centriole length by CPAP and CP110. Curr. Biol. 19, 1005–1011 10.1016/j.cub.2009.05.016 PubMed DOI

Zhang Y., Seo S., Bhattarai S., Bugge K., Searby C. C., Zhang Q., Drack A. V., Stone E. M., and Sheffield V. C. (2014) BBS mutations modify phenotypic expression of CEP290-related ciliopathies. Hum. Mol. Genet. 23, 40–51 10.1093/hmg/ddt394 PubMed DOI PMC

Tsang W. Y., Bossard C., Khanna H., Peränen J., Swaroop A., Malhotra V., and Dynlacht B. D. (2008) CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease. Dev. Cell 15, 187–197 10.1016/j.devcel.2008.07.004 PubMed DOI PMC

Seo S., Baye L. M., Schulz N. P., Beck J. S., Zhang Q., Slusarski D. C., and Sheffield V. C. (2010) BBS6, BBS10, and BBS12 form a complex with CCT/TRiC family chaperonins and mediate BBSome assembly. Proc. Natl. Acad. Sci. U. S. A. 107, 1488–1493 10.1073/pnas.0910268107 PubMed DOI PMC

Stoetzel C., Muller J., Laurier V., Davis E. E., Zaghloul N. A., Vicaire S., Jacquelin C., Plewniak F., Leitch C. C., Sarda P., Hamel C., de Ravel T. J., Lewis R. A., Friederich E., Thibault C., et al. (2007) Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome. Am. J. Hum. Genet. 80, 1–11 10.1086/510256 PubMed DOI PMC

Stoetzel C., Laurier V., Davis E. E., Muller J., Rix S., Badano J. L., Leitch C. C., Salem N., Chouery E., Corbani S., Jalk N., Vicaire S., Sarda P., Hamel C., Lacombe D., et al. (2006) BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus. Nat. Genet. 38, 521–524 10.1038/ng1771 PubMed DOI

Katsanis N., Beales P. L., Woods M. O., Lewis R. A., Green J. S., Parfrey P. S., Ansley S. J., Davidson W. S., and Lupski J. R. (2000) Mutations in MKKS cause obesity, retinal dystrophy and renal malformations associated with Bardet-Biedl syndrome. Nat. Genet. 26, 67–70 10.1038/79201 PubMed DOI

Labun K., Montague T. G., Krause M., Torres Cleuren Y. N., Tjeldnes H., and Valen E. (2019) CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 10.1093/nar/gkz365 PubMed DOI PMC

Ran F. A., Hsu P. D., Wright J., Agarwala V., Scott D. A., and Zhang F. (2013) Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 10.1038/nprot.2013.143 PubMed DOI PMC

Schwille P., Haupts U., Maiti S., and Webb W. W. (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77, 2251–2265 10.1016/S0006-3495(99)77065-7 PubMed DOI PMC

Widengren J., Mets U., and Rigler R. (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J. Phys. Chem. 99, 13368–13379 10.1021/j100036a009 DOI

Machán R., and Hof M. (2016) Fluorescence Correlation Spectroscopy (FCS). in Practical Manual for Fluorescence Microscopy Techniques (Ahmed S., Thankiah S., Machan R., Hof M., Clayton A. H. A., Wright G., Sibarita J.-B., Korte T., and Herrmann A., eds) p. 5–9, PicoQuant GmbH, Berlin

BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

A protocol for generation and live-cell imaging analysis of primary cilia reporter cell lines

The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly