The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells
2020; Elsevier BV; Volume: 295; Issue: 42 Linguagem: Inglês
10.1074/jbc.ra120.013905
ISSN1083-351X
AutoresAvishek Prasai, Markéta Schmidt Černohorská, Klara Ruppova, Veronika Niederlová, Monika Andelova, Peter Dráber, Ondřej Štěpánek, Martina Huranová,
Tópico(s)Renal and related cancers
ResumoBardet–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. 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. Bardet–Biedl Syndrome (BBS) is a multiorgan genetic disorder caused by the dysfunction of the primary cilia, microtubule-based sensory organelles. BBS is primarily characterized by retinopathy, polydactyly, genital and renal anomalies, obesity, and cognitive impairment (1Forsythe E. Beales P.L. Bardet-Biedl syndrome.Eur. J. Hum. Genet. 2013; 21 (22713813): 8-1310.1038/ejhg.2012.115Crossref PubMed Scopus (291) Google Scholar). Twenty-four causative BBS genes have been identified so far, eight of which form a stable protein complex called the BBSome (2Niederlova V. Modrak M. Tsyklauri O. Huranova M. Stepanek O. Meta-analysis of genotype-phenotype associations in Bardet-Biedl syndrome uncovers differences among causative genes.Hum. Mutat. 2019; 40 (31283077): 2068-208710.1002/humu.23862Crossref PubMed Scopus (21) Google Scholar, 3Nachury 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. Jackson P.K. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.Cell. 2007; 129 (17574030): 1201-121310.1016/j.cell.2007.03.053Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). The BBSome is an adaptor protein complex consisting of BBS1, -2, -4, -5, -7, -8, -9, and -18, possessing structural similarities to coat/adaptor proteins involved in vesicular trafficking (3Nachury 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. Jackson P.K. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.Cell. 2007; 129 (17574030): 1201-121310.1016/j.cell.2007.03.053Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 4Loktev 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. Nachury M.V. A BBSome subunit links ciliogenesis, microtubule stability, and acetylation.Dev. Cell. 2008; 15 (19081074): 854-86510.1016/j.devcel.2008.11.001Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The BBSome works in concert with the intraflagellar transport machinery to facilitate the trafficking of particular transmembrane cargoes into and out of the primary cilia (5Wingfield J.L. Lechtreck K.F. Lorentzen E. Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery.Essays Biochem. 2018; 62 (30287585): 753-76310.1042/EBC20180030Crossref PubMed Scopus (42) Google Scholar, 6Dilan T.L. Singh R.K. Saravanan T. Moye A. Goldberg A.F.X. Stoilov P. Ramamurthy V. Bardet-Biedl syndrome-8 (BBS8) protein is crucial for the development of outer segments in photoreceptor neurons.Hum. Mol. Genet. 2018; 27 (29126234): 283-29410.1093/hmg/ddx399Crossref PubMed Scopus (28) Google Scholar, 7Nachury M.V. The molecular machines that traffic signaling receptors into and out of cilia.Curr. Opin. Cell Biol. 2018; 51 (29579578): 124-13110.1016/j.ceb.2018.03.004Crossref PubMed Scopus (61) Google Scholar, 8Liu P.W. Lechtreck K.F. 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Genet. 2012; 21 (22228099): 1945-195310.1093/hmg/dds004Crossref PubMed Scopus (94) Google Scholar), leptin signaling (11Guo D.F. Cui H. Zhang Q. Morgan D.A. Thedens D.R. Nishimura D. Grobe J.L. Sheffield V.C. Rahmouni K. The BBSome controls energy homeostasis by mediating the transport of the leptin receptor to the plasma membrane.PLoS Genet. 2016; 12 (26926121): e100589010.1371/journal.pgen.1005890Crossref PubMed Scopus (55) Google Scholar), photoreceptor signaling (12Nishimura 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. Sheffield V.C. 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. 2004; 101 (15539463): 16588-1659310.1073/pnas.0405496101Crossref PubMed Scopus (279) Google Scholar), and neuronal signaling by G protein–coupled receptors (13McIntyre J.C. Hege M.M. Berbari N.F. Trafficking of ciliary G protein-coupled receptors.Methods Cell Biol. 2016; 132 (26928538): 35-5410.1016/bs.mcb.2015.11.009Crossref PubMed Scopus (17) Google Scholar, 14Loktev A.V. Jackson P.K. Neuropeptide Y family receptors traffic via the Bardet-Biedl syndrome pathway to signal in neuronal primary cilia.Cell Rep. 2013; 5 (24316073): 1316-132910.1016/j.celrep.2013.11.011Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Berbari N.F. Lewis J.S. Bishop G.A. Askwith C.C. Mykytyn K. Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia.Proc. Natl. Acad. Sci. U. S. A. 2008; 105 (18334641): 4242-424610.1073/pnas.0711027105Crossref PubMed Scopus (320) Google Scholar). Mutations in any of the BBSome subunits can cause BBS, suggesting that every subunit is essential for complete BBSome function (2Niederlova V. Modrak M. Tsyklauri O. Huranova M. Stepanek O. Meta-analysis of genotype-phenotype associations in Bardet-Biedl syndrome uncovers differences among causative genes.Hum. Mutat. 2019; 40 (31283077): 2068-208710.1002/humu.23862Crossref PubMed Scopus (21) Google Scholar). The structure of the BBSome was a long-standing enigma until recently, because the indirect approaches, such as the yeast two-hybrid system (16Woodsmith J. Apelt L. Casado-Medrano V. Özkan Z. Timmermann B. Stelzl U. Protein interaction perturbation profiling at amino-acid resolution.Nat. Methods. 2017; 14 (29039417): 1213-122110.1038/nmeth.4464Crossref PubMed Scopus (19) Google Scholar), co-precipitation (17Katoh Y. Nozaki S. Hartanto D. Miyano R. Nakayama K. Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins.J. Cell Sci. 2015; 128 (25964651): 2351-236210.1242/jcs.168740Crossref PubMed Scopus (71) Google Scholar), co-expression of the individual subunits followed by low-resolution cryo-EM (18Klink B.U. Zent E. Juneja P. Kuhlee A. Raunser S. Wittinghofer A. A recombinant BBSome core complex and how it interacts with ciliary cargo.Elife. 2017; 6 (29168691): e2743410.7554/eLife.27434Crossref PubMed Scopus (42) Google Scholar), and structural analysis of individual BBSome subunits (19Mourão A. Nager A.R. Nachury M.V. Lorentzen E. Structural basis for membrane targeting of the BBSome by ARL6.Nat. Struct. Mol. Biol. 2014; 21 (25402481): 1035-104110.1038/nsmb.2920Crossref PubMed Scopus (47) Google Scholar, 20Knockenhauer K.E. Schwartz T.U. Structural characterization of Bardet-Biedl syndrome 9 protein (BBS9).J. Biol. Chem. 2015; 290 (26085087): 19569-1958310.1074/jbc.M115.649202Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), provided only a very limited insight into the overall BBSome structure. Moreover, some authors proposed that BBS2, -7, and -9 form the BBSome core enabling the subsequent recruitment of other subunits (21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome.J. Biol. Chem. 2012; 287 (22500027): 20625-2063510.1074/jbc.M112.341487Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), whereas others showed that BBS2 and BBS7 are dispensable for the assembly of the remaining six subunits (18Klink B.U. Zent E. Juneja P. Kuhlee A. Raunser S. Wittinghofer A. A recombinant BBSome core complex and how it interacts with ciliary cargo.Elife. 2017; 6 (29168691): e2743410.7554/eLife.27434Crossref PubMed Scopus (42) Google Scholar, 22Klink B.U. Gatsogiannis C. Hofnagel O. Wittinghofer A. Raunser S. Structure of the human BBSome core complex.Elife. 2020; 9 (31951201): e5391010.7554/eLife.53910Crossref PubMed Scopus (16) Google Scholar). Three independent studies resolved the BBSome structure using cryo-EM with molecular modeling using the BBSome isolated from bovine retina (23Chou 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.The molecular architecture of native BBSome obtained by an integrated structural approach.Structure. 2019; 27 (31303482): 1384-1394.e410.1016/j.str.2019.06.006Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) or using high-resolution cryo-EM analysis of bovine BBSome (24Singh S.K. Gui M. Koh F. Yip M.C. Brown A. Structure and activation mechanism of the BBSome membrane protein trafficking complex.Elife. 2020; 9 (31939736): e5332210.7554/eLife.53322Crossref PubMed Scopus (14) Google Scholar) or of human BBSome (lacking BBS2 and BBS7) expressed in insect cells (22Klink B.U. Gatsogiannis C. Hofnagel O. Wittinghofer A. Raunser S. Structure of the human BBSome core complex.Elife. 2020; 9 (31951201): e5391010.7554/eLife.53910Crossref PubMed Scopus (16) Google Scholar). These studies showed a high level of interconnectivity among the BBSome subunits, raising the question of how these BBSome proteins assemble in the cells. Quantitative fluorescence microscopy techniques can reveal the formation and dynamics of functional protein complexes in living cells (25Huranová M. Ivani I. Benda A. Poser I. Brody Y. Hof M. Shav-Tal Y. Neugebauer K.M. Stanek D. The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells.J. Cell Biol. 2010; 191 (20921136): 75-8610.1083/jcb.201004030Crossref PubMed Scopus (74) Google Scholar, 26Huranova M. Muruganandam G. Weiss M. Spang A. Dynamic assembly of the exomer secretory vesicle cargo adaptor subunits.EMBO Rep. 2016; 17 (26742961): 202-21910.15252/embr.201540795Crossref PubMed Scopus (11) Google Scholar). In this study, we applied multiple microscopy and biochemical techniques to a large library of genetically engineered human RPE1 cells to describe spatially resolved steps of BBSome formation in cells. To study BBSome formation in cells, we initially established stable WT RPE1 cell lines expressing BBSome subunits fused with super yellow fluorescent protein SYFP2 (YFP) (27Kremers G.J. Goedhart J. van Munster E.B. Gadella Jr., T.W. Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius.Biochemistry. 2006; 45 (16716067): 6570-658010.1021/bi0516273Crossref PubMed Scopus (292) Google Scholar). BBS1, BBS4, BBS8, and BBS18 were tagged at the N termini, whereas the BBS7 and BBS9 were tagged at C termini, because the N-terminal tag interfered with their function (data not shown). All YFP-tagged BBSome subunits localized to the primary cilia of WT RPE1 cells and showed weak diffuse signals throughout the cytoplasm (Fig. 1A). The pattern of the diffused cytoplasmic localization of the BBSome subunits was not affected by the cell density. For all the following experiments, the cells were kept 80–100% confluent. YFP-BBS4 and YFP-BBS18 additionally localized to the pericentriolar satellites (PS) around the basal body as shown before (Fig. 1A) (28Kim 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. Beales P.L. The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression.Nat. Genet. 2004; 36 (15107855): 462-47010.1038/ng1352Crossref PubMed Scopus (324) Google Scholar, 29Chamling X. Seo S. Searby C.C. Kim G. Slusarski D.C. Sheffield V.C. The centriolar satellite protein AZI1 interacts with BBS4 and regulates ciliary trafficking of the BBSome.PLoS Genet. 2014; 10 (24550735): e100408310.1371/journal.pgen.1004083Crossref PubMed Scopus (22) Google Scholar). In the next step, we generated RPE1 cell lines deficient in BBS1, BBS2, BBS4, BBS7, TTC8/BBS8, BBS9, or BBIP1/BBS18 using the CRISPR/Cas9 technology (Table S1). Each of the seven BBS-deficient cell lines was subsequently transduced with retroviral vectors to express YFP-tagged BBS1, -4, -5, -7, -8, -9, or -18, giving rise to a library of 63 stable cell lines in total (Table S2). Lack of any of the BBSome subunits prevented the ciliary localization of YFP-tagged BBS1, BBS4, BBS5, BBS7, BBS8, BBS9, and BBS18 (Fig. S1). Likewise, the endogenous BBS9 was rather diffused throughout the cytoplasm in the KO cells, whereas it localized to primary cilia in WT cells and KO cells reconstituted with the missing YFP-tagged subunits (e.g. YFP-BBS1 in BBS1 KO) (Fig. S2A). These data suggested that only the intact BBSome, and not BBSome intermediates or subunits alone, could enter cilia. Despite substantial overexpression of the YFP-tagged subunits over their endogenous counterparts (Fig. S2B), the fluorescence signal was still rather weak and mostly comparable in the respective WT and KO cell lines (Figs. S1 and S2C). Cells deficient in BBS1, BBS4, BBS5, BBS7, BBS9, or BBS18 formed significantly shorter cilia compared with WT cells (Fig. 1B), corresponding to a previous observation in BBS4-deficient cells (30Hernandez-Hernandez V. Pravincumar P. Diaz-Font A. May-Simera H. Jenkins D. Knight M. Beales P.L. Bardet-Biedl syndrome proteins control the cilia length through regulation of actin polymerization.Hum. Mol. Genet. 2013; 22 (23716571): 3858-386810.1093/hmg/ddt241Crossref PubMed Scopus (67) Google Scholar). This phenotype was rescued by expressing YFP-tagged variants of the missing genes (Fig. 1B), documenting both that the cilia shortening was indeed caused by the gene deficiencies and that the YFP-tag does not interfere with the function of the BBSome subunits. Intriguingly, deletion of BBS8 resulted in prolonged cilia, which was reverted by the expression of YFP-BBS8 (Fig. 1C). The unexpected phenotype of the BBS8 KO cell line could be caused by a unique role of BBS8 or could be an artifact of the particular mutation. Deficiency of any BBSome subunit reduced the endogenous levels of the other subunits in RPE1 KO cell lines (Fig. S3 (A and B) and Table S1), indicating that the BBSome and/or BBSome intermediates are more stable than free individual subunits. In particular, deficiency in any of the subunits forming the proposed core of the BBSome (i.e. BBS2, BBS7, or BBS9) (21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome.J. Biol. Chem. 2012; 287 (22500027): 20625-2063510.1074/jbc.M112.341487Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) substantially reduced the cellular levels of other subunits, whereas the absence of BBS1, BBS4, BBS8, or BBS18 had less dramatic effects on the stability of other subunits. The interdependence of the individual BBSome subunits suggests that they are present predominantly in the form of the BBSome or BBSome intermediates in WT cells. We addressed whether ciliogenesis (stimulated by serum starvation) is coupled with de novo formation of the BBSome or whether the BBSome is preformed in nonciliated cells. BBS1, -2, -5, -7, -8, and -9 co-immunoprecipitated with YFP-BBS4 in the nonstarved cells (Fig. 2A). However, the serum starvation increased the amount of co-precipitated BBSome subunits ∼2–4-fold, indicating that the BBSome formation is slightly augmented upon induction of ciliogenesis in RPE1 cells (Fig. 2 (A and B) and Fig. S4A). Under these conditions, only 5% of cells formed cilia, which suggested that the BBSome assembles also in the nonciliated cells (Fig. S4A). The presence of the BBSome in nonciliated cells suggests that the BBSome or BBSome intermediates are present in the cytoplasm. Using fluorescence correlation spectroscopy (FCS), we estimated the diffusion speed of YFP-tagged subunits in WT and KO cell lines (Table S3). Because the large proteins and complexes diffuse slower than small complexes, these measurements reveal the information about the relative size of the respective complexes. We observed that the diffusion speed of YFP-BBS4 was significantly faster in BBS9 KO cells than in WT cells, providing evidence that a fraction of BBS4 resides in a BBS9-dependent complex in the cytoplasm (Fig. 2, C and F). We performed a two-component fitting of the WT data and found that the diffusion speed of this complex is about 10 times slower than the free YFP-BBS4 (Fig. 2C). Significant but less pronounced effects were observed in BBS1 KO and BBS7 KO (Fig. 2, D–F). These results show that cytoplasmic BBS4 exists in a form of a complex with BBS9 and likely other BBSome subunits. YFP-tagged BBSome subunits, other than BBS4, showed significant differences in their mobility between WT and KO cells only in some rare cases (Fig. S4, B–E). It is possible that their overexpression masked the effect of BBSome disruption by favoring the monomeric forms (Fig. S3, B and C). In the case of YFP-BBS1, we could observe a mild loss in the slower fraction in BBS7 or BBS9 KO cells (Fig. S4C). Overall, these data imply the existence of heterogenic complexes of BBSome subunits in the cytoplasm. BBS4 interacts with PCM-1 (pericentriolar material-1) (28Kim 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. Beales P.L. The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression.Nat. Genet. 2004; 36 (15107855): 462-47010.1038/ng1352Crossref PubMed Scopus (324) Google Scholar), which leads to its enrichment at the PS in both ciliated and nonciliated cells (Fig. S5A). BBS9 localizes to PS as well, albeit to a much lesser extent than BBS4 (Fig. 3, A and B), indicating that a fraction of BBS9 and potentially other BBSome subunits reside at the PS in starved WT cells. Surprisingly, in BBS1 KO cells, both the BBS4 and BBS9 were highly enriched at the PS (Fig. 3, A and B). In addition, we observed that all other BBSome subunits (i.e. YFP-tagged BBS5, BBS7, BBS8, BBS9, and BBS18 and endogenous BBS2 and BBS9) localize to PS in BBS1 KO cells (Fig. 3C and Fig. S5B). The enrichment of the BBSome subunits at the PS depended on BBS4, because it was not observed in BBS4-deficient cells and BBS1/BBS4 DKO cells (Fig. 3D). To analyze the localization of BBS9 with higher resolution, we employed expansion microscopy (31Gambarotto 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. Guichard P. Imaging cellular ultrastructures using expansion microscopy (U-ExM).Nat. Methods. 2019; 16 (30559430): 71-7410.1038/s41592-018-0238-1Crossref PubMed Scopus (99) Google Scholar). In line with the previous observations, BBS9 localized to the cilium in WT cells, localized to PS in BBS1 KO cells and was absent from the ciliary/centrosomal proximity in BBS4 KO cells (Fig. 3E). Fluorescence recovery after photobleaching (FRAP) analysis revealed comparable recovery half-times for all YFP-tagged BBSome subunits in BBS1 KO cells at the PS (Fig. 3 (F and G) and Table S4), possibly because they are predominantly engaged in a single complex. YFP-BBS4 showed the highest immobile fraction, which is consistent with its direct binding to the PS via PCM-1 (Fig. 3F). Most likely, BBS4 recruits other subunits to the PS, leading to the formation of a pre-BBSome complex. The accumulation of the BBSome intermediates at the PS in BBS1 KO cells results in the mobilization of BBS4 (Fig. 3 (H and I) and Table S4), indicating that the pre-BBSome binding to PS is less stable than binding of BBS4 alone. BBS1 is crucial for the completion of the full BBSome, which prevents the BBSome subunits, with the exception of BBS4, from the arrest at the PS. As all BBSome subunits, BBS1 localizes to the cilia in WT cells (Fig. 1A). However, we observed also quite frequent BBS1 abundance at the centrosome in nonciliated cells and at the centrosome/basal body in some cells with short nascent cilia (Fig. 4A). We calculated that BBS1 localizes to the centrosome in ∼80% nonciliated cells, whereas the other BBSome subunits localize only in 10–30% nonciliated cells (Fig. S5C). Moreover, BBS1 localizes to the centrosome/basal body in BBS4 KO cells, whereas other BBSome subunits show diffuse localization in the cytoplasm of these cells (Fig. 4B). Deficiency of BBS4 increased the turnover of BBS1 at the centrosome/basal body (Fig. 4, C and D), suggesting that the interaction of BBS1 with the pre-BBSome stabilizes BBS1 at the centrosome/basal body, possibly directing the whole complex toward the cilium. We performed an analysis of the dynamic behavior of the BBSome subunits at the ciliary tip and the basal body and the transition zone using FRAP (Fig. 5 (A–C) and Table S5). If the subunits are incorporated in the whole BBSome quantitatively, their recovery rate should be comparable. However, BBS1 behaved differently from the other subunits, because it was very mobile at the base of the cilium (Fig. 5C and Fig. S5E). Indeed, BBS1 was the only subunit that was more dynamic in the ciliary base than in the ciliary tip (Fig. 5D and Fig. S5 (D and E)). These data indicate that BBS1 exists in two forms at the ciliary base, as a monomeric protein with fast turnover between the ciliary base and the cytoplasm and as a part of the BBSome, whereas other BBSome subunits are recruited to the ciliary base only in the form of the complete BBSome complex. Interestingly, the absence of BBS1 not only stalls BBSome at the PS, but alters the structure of the ciliary base, resulting in a slightly prolonged basal body and transition zone (Fig. 5, E and F), suggesting a role of the BBS1 in the organization of the ciliary base. Based on our data, we propose a model of the BBSome formation in cells with the main roles for BBS4 and BBS1 in the spatial regulation of the full complex assembly (Fig. 6). BBS4 resides on PS, and via its association with BBS9, it recruits other BBSome subunits to form the pre-BBSome complex. BBS1 localizes to the basal body and guides the pre-BBSome to the ciliary base, where it facilitates the BBSome translocation to the cilium. Because the BBSome consists of eight subunits, it has been proposed that it assembles in a stepwise manner (21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome.J. Biol. Chem. 2012; 287 (22500027): 20625-2063510.1074/jbc.M112.341487Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, in vitro experiments provided contradictory data concerning the sequence of the individual steps (18Klink B.U. Zent E. Juneja P. Kuhlee A. Raunser S. Wittinghofer A. A recombinant BBSome core complex and how it interacts with ciliary cargo.Elife. 2017; 6 (29168691): e2743410.7554/eLife.27434Crossref PubMed Scopus (42) Google Scholar, 21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome.J. Biol. Chem. 2012; 287 (22500027): 20625-2063510.1074/jbc.M112.341487Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 22Klink B.U. Gatsogiannis C. Hofnagel O. Wittinghofer A. Raunser S. Structure of the human BBSome core complex.Elife. 2020; 9 (31951201): e5391010.7554/eLife.53910Crossref PubMed Scopus (16) Google Scholar). Although the resolution of the structure of the BBSome complex represented a breakthrough in the field (22Klink B.U. Gatsogiannis C. Hofnagel O. Wittinghofer A. Raunser S. Structure of the human BBSome core complex.Elife. 2020; 9 (31951201): e5391010.7554/eLife.53910Crossref PubMed Scopus (16) Google Scholar, 23Chou 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.The molecular architecture of native BBSome obtained by an integrated structural approach.Structure. 2019; 27 (31303482): 1384-1394.e410.1016/j.str.2019.06.006Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 24Singh S.K. Gui M. Koh F. Yip M.C. Brown A. Structure and activation mechanism of the BBSome membrane protein trafficking complex.Elife. 2020; 9 (31939736): e5332210.7554/eLife.53322Crossref PubMed Scopus (14) Google Scholar), these studies could not reveal how the BBSome assembles in living cells, including the spatial regulation of individual steps. We generated a library of 64 RPE1-derived cell lines using a combination of CRISPR/Cas9 knockouts of individual BBSome subunits and retroviral expression of YFP-tagged subunits. The only misses in the library were BBS5 KO and BBS2-YFP, because we were unsuccessful in their preparation. We used these lines to address the localization of individual BBSome subunits in WT cells and in cells lacking another subunit. The signal from YFP-tagged proteins was complemented with the detection of endogenous BBS9 and BBS2 using immunofluorescence. Moreover, we used two microscopy techniques, FCS and FRAP, to elucidate the dynamics of individual subunits in our cell lines. Altogether, our data enabled us to propose a model of BBSome formation in living cells. It has been shown that some BBSome subunits stabilize each other in cells (21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet-Biedl syndrome protein complex, the BBSome.J. Biol. Chem. 2012; 287 (22500027): 20625-2063510.1074/jbc.M112.341487Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 32Zhang Q. Nishimura D. Vogel T. Shao J. Swiderski R. Yin T. Searby C. Carter C.S. Kim G. Bugge K. Stone E.M. Sheffield V.C. 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. 2013; 126 (23572516): 2372-238010.1242/jcs.111740Crossref PubMed Scopus (71) Google Scholar). Our systematic analysis demonstrated that actually all BBSome subunits are interdependent. Deficiency in any of the subunits (with the exception of BBS5, which was not tested) leads to the substantial decrease of the cellular levels of other subunits, indicating that only a minor fraction of the BBSome subunit molecules are monomeric. Deficiency of BBS2, BBS7, or BBS9 had the most pronounced effects on the abundance of other subunits, suggesting that these three structurally related proteins form the core of the BBSome in vivo (21Zhang Q. Yu D. Seo S. Stone E.M. Sheffield V.C. Intrinsic protein-protein interaction-medi
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