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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01676

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MKKS/BBS6, a divergent chaperonin-like protein linked to the obesity disorder Bardet-Biedl syndrome, is a novel centrosomal component required for cytokinesis

¹ Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada
² Department of Cell Biology and Anatomy, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada
³ Institute of Genetic Medicine and Wilmer Eye Institute, Johns Hopkins University, North Wolfe Street, Baltimore, MD 21287, USA
⁴ Molecular Medicine Unit, Institute of Child Health, University College London, London, WC1 1EH, UK
⁵ Department of Biochemistry and Molecular Biology, Dalhousie University, 5850 College Street, Halifax, NS, B3H 1X5, Canada

View larger version (35K): [in a new window]   Fig. 1. BBS6 is a highly divergent eukaryotic Group II chaperonin related to CCT. (A) Maximum likelihood phylogenetic tree constructed from an alignment containing 5 BBS6 sequences, 11 archaeal chaperonins and 32 eukaryotic CCTs (four from each of the eight different CCT subunits). The BBS6 sequences are highlighted, and PROML bootstrap support values are provided for significant nodes on the tree where they are >50%. The BBS6 proteins show a weakly supported but consistently observed relationship with the subunit of CCT, suggesting that the gene encoding BBS6 is a divergent, duplicated CCT subunit. The scale bar indicates the estimated number of amino acid substitutions per site. Abbreviations: Cren; Crenarchaeotes, Eur; Euryarchaeotes. (B) Amino acid identities shared between Homo sapiens, Mus musculus, Danio rerio and Ciona intestinalis BBS6 and CCT protein sequences. (C) BBS6 proteins share signature sequences that differ from other chaperonins. Alignments of two sequences from human BBS6 (HsBBS6), Danio BBS6 (DrBBS6) and Ciona BBS6 (CiBBS6) are compared to a consensus sequence derived from CCT and archaeal chaperonins (see Fig. S1 in supplementary material). Positions known to interact with the different regions of the ATP moiety (Ditzel et al., 1998) are indicated with inverted triangles under the alignments.

View larger version (42K): [in a new window]   Fig. 2. BBS6 is enriched in centrosomal fractions and its minor soluble form does not associate with CCT or oligomerize. (A) BBS6 is highly enriched in centrosomes purified from NIH 3T3 cells. HeLa cells were either not transfected or transfected with pCMV-Myc-BBS6 or pCMV-BBS6, and total cell lysates were probed by western blot analysis with an affinity-purified polyclonal BBS6 antibody. Wild-type (60 kDa) and myc-tagged BBS6 (62 kDa) can be observed in the transfected cells. (B) Most of BBS6 occurs as a high-MW Triton X-insoluble form distinct from that of cytosolic CCT. Total lysates of IMCD3 cells expressing pCMV-BBS6 were loaded on discontinuous 4-71% sucrose gradient (the top, bottom and sucrose steps in the gradient are indicated). After centrifugation, each fraction was analyzed on a 10% SDS polyacrylamide gel and subjected to western blot analyses with antibodies to BBS6, CCT or -tubulin, as indicated. Most of the BBS6 and -tubulin is found in the 71% and pellet fractions, while the majority of CCT forms a peak at 24% sucrose. Schematics below the panels represent potential positions of monomeric/dimeric or oligomeric chaperonin species. (C) BBS6 does not form homo-oligomers. Extracts of COS-7 cells transiently expressing different constructs (i.e. pEGFP-BBS6 or pCMV-Myc-BBS6, as indicated), or untransfected, were immunoprecitated (IP) with antibodies to myc, GFP or CCT. Lysates (L), immune supernatants (S) or immune pellets (P) were subjected to western blot analyses using antibodies against BBS6, myc, GFP or CCT (UM1 antibody), as indicated. The respective positions of myc-BBS6, GFP-BBS6 and CCT subunits are noted.

View larger version (86K): [in a new window]   Fig. 3. BBS6 is a centrosomal protein that is also found at the midbody during cytokinesis. (A) Two discrete perinuclear signals are observed in IMCD3 cells by immunocytochemistry using an affinity-purified anti-BBS6 polyclonal antibody. (B) Immunolocalization studies of endogenous BBS6 (top panels) and transiently expressed myc-tagged, and GFP-tagged BBS6 (middle and bottom panels, respectively) show that BBS6 colocalizes with the centrosomal marker, -tubulin. The transfected cells were fixed and costained with antibodies to myc and -tubulin, and GFP-BBS6 signal was detected by direct GFP fluorescence (middle and bottom panels). Left, middle and right panels, respectively, show BBS6 (green), -tubulin (red) and a merge of the two signals (yellow indicates overlap). (C) Endogenous BBS6 is found at both basal bodies (mother centrioles, displaying a primary cilium) and daughter centrioles. BBS6 is stained green and cilia are stained red with anti-acetylated-tubulin antibodies. (D) BBS6 remains associated with centrosomes throughout the cell cycle. Synchronized IMCD3 cells were co-stained with antibodies to BBS6 (green) and -tubulin (red); yellow indicates overlapping signals. (E) BBS6 is found at the centrosomes (indicated by arrowheads) and midbody region (indicated by an arrow) during telophase, as judged by its colocalization with -tubulin.

View larger version (70K): [in a new window]   Fig. 4. BBS6 is localized within the pericentriolar material (PCM) tube and shows a dynamic distribution during the cell cycle. (A) High-resolution digital microscopy optical sections of NIH 3T3 cells co-stained with antibodies against BBS6 (green) and - and acetylated-tubulin (red). (B) Optical sections of BBS6 (green) and PCM tube (red) signals in the same focal plane from interphase or mitotic HeLa cells. The PCM tube was detected using an antibody, M4491, that recognizes multiple PCM components. Closeups of the PCM tube (insets), BBS6 and merged signals are also shown.

View larger version (102K): [in a new window]   Fig. 5. Centrosomal assembly of BBS6 is not dependent on polymerized microtubules or the dynein-dynactin molecular motor. (A) IMCD3 cells were either mock-treated (no drug) or treated with nocodazole for 1 hour and allowed to recover for 0 or 10 minutes. Cells were then co-stained with antibodies to BBS6 or -tubulin (green) and -tubulin (red). (B) IMCD3 cells were transiently transfected with myc-tagged p50-dynamitin, which inhibits dynein-dynactin motor function. Cells were then stained with an anti-myc antibody (red) and costained with antibodies to BBS6, PCM1 or -tubulin (green). Arrows denote centrosomal BBS6 and -tubulin signals from p50-dynamitin-transfected cells, and arrowheads denote centrosomal BBS6, PCM1 and -tubulin in untransfected cells. Note the dispersed PCM1 signal in transfected cells.

View larger version (91K): [in a new window]   Fig. 6. The centrosomal localization of BBS6 is abrogated by several mutations found in patients, and is conferred by the apical domain region. (A) Schematics of the BBS6 variants tested. The equatorial, intermediate and apical domain regions are indicated, along with the positions of the six missense variants, and the extent of the truncation in the delGG mutant. (B) Western blot analysis of all GFP-tagged BBS6 variants expressed in COS-7 cells, as detected with an anti-GFP antibody. (C) Localization of GFP-tagged BBS6 variants detected via GFP fluorescence. Two mutants (R156L and C499S) localize to the centrosome, as with GFP-tagged wild-type (WT) BBS6 (WT: 69% of the cells show centrosomal localization, n=190, R156L: 53%, n=130, C499S: 72%, n=130, top panels); four variants fail to localize to the centrosome (G52D: 2% of the cells show centrosomal localization, n=155, G345E: 1%, n=100, D285A: 7%, n=150 and T325P: 0%, n=128; bottom panels). (D) The GFP-tagged apical domain of BBS6 localizes to the centrosome, as does the delGG truncation mutant (apical: 46% of the cells show centrosomal localization, n=144, delGG: 34%, n=200).

View larger version (86K): [in a new window]   Fig. 7. Silencing of BBS6 by RNAi in COS-7 and NIH 3T3 cells produces multi-nucleated and multi-centrosomal cells, and cytokinesis defects. (A) DIC images of COS-7 and NIH 3T3 cells 9 days after transfection with BBS6 siRNA expression vector (pSilencer-BBS6 or pSuper-BBS6, respectively) show interconnected cells with long intercellular bridges as a result of defective cytokinesis. Immunocytochemistry with a -tubulin antibody (red) and DAPI staining of nuclei (blue) show that BBS6 silencing causes bi-nucleated and multi-centrosomal phenotypes in both COS-7 and NIH 3T3 cells. The merged images (right panels) show two nuclei in single cells. (B) Microtubule organization is not significantly altered during cell cycle progression in COS-7 cells transfected with pSilencer-BBS6 (upper panels) compared with cells transfected with pSilencer empty vector (lower panels). Cells were stained with antibodies to -tubulin (red), -tubulin (green) and counterstained with DAPI (blue). Only merged images are shown; yellow indicates overlapping - and -tubulin signals.

View larger version (63K): [in a new window]   Fig. 8. BBS6 is enriched in organs affected in BBS and in ciliated cells. (A) Wholemount RNA in situ hybridization of an E10 mouse embryo showing strong BBS6 expression in the forebrain (Fb), retina (Re), branchial arches (Ba), developing heart (He) and limb bud (Lb). (B) Immunohistochemistry using BBS6 antibody; saggital section of a mouse E13.5 brain showing protein staining in the developing nervous system, including the neopallial cortex (Nc), the roof of the midbrain (Rm), the cerebellar primordium (Ce), the choroid plexus (Cp), the hypothalamus (Hy) and Rathke's pouch (Rp). Modest BBS6 protein staining is also detectable in the thalamus (Th) and the medulla oblongata (Mo). (C) In the adult kidney, BBS6 localizes to the ciliated border of renal tubular cells (Rtc). (D) In the adult photoreceptor, BBS6 is detected in the connecting cilium (Cc) and the inner and outer nuclear layers (Inl, Onl) but not the outer or inner segment (Os, Is). (E,F) BBS6 localizes to the ciliated layer of the olfactory epithelium (cl) as well as the ependymal layer (El) of ventricles (Ve); Lu, lumen. (G) A section of mouse testis is stained for BBS6 (green) and the PCM tube (antibody M4491; red). The area shown is taken from the periphery of a semineferous tubule containing both meiotic and spermatogenic cells. BBS6 colocalizes to centrosomes/basal bodies, but not the flagellar axoneme, in spermatogenic cells (see arrowheads). Also shown are nuclei (DAPI stained, blue) and a merged image.

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