Skip to main content
. 2011 Nov 25;21(6):1272–1286. doi: 10.1093/hmg/ddr557

Figure 1.

Figure 1.

Meckelin localizes to the primary cilium and basal body and interacts with filamin. (A) Domain structure of meckelin showing the locations of predicted signal peptide, cysteine-rich repeat region, a region containing potential three to seven transmembrane domains (TM) and a coiled-coil domain. Indicated are the locations of epitopes for two anti-meckelin antibodies (‘Nt Ab’ and ‘Ct Ab’), the C-terminal region used for a Y2H bait fragment and a meckelin Ct-GFP construct, and the pathogenic MKS in-frame deletion mutation p.919delF (red). Numbering indicates the amino-acid residue. (B) Upper panels: Co-immunostaining and confocal microscopy of a ciliated mouse IMCD3 epithelial cell for meckelin (using the Nt Ab; green in merged image) and acetylated (Ac)-α-tubulin antibody (red) showing co-localization of meckelin isoforms at the cilium. A confocal x–y projection (planar projn.) and x–z-side projection are shown. Enlarged insets (white frames) show detail of localization at the proximal region of the cilium (arrow) and the basal body (barbed arrow). Nuclear localization of 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. Scale bar, 5 µm. Lower panels: 1 μm confocal section of the apical cell surface following co-immunostaining for meckelin Ct (green) and γ-tubulin (red). At the apical surface, meckelin had a punctate distribution and strong peribasal body accumulation. Scale bar, 5 µm. (C) Domain structure of filamin A showing meckelin-binding domain, actin-binding domain, dimerization domain and hinge. Numbering indicates the amino-acid residue. (D) Left panel: IP of endogenous filamin A (size 280 kDa) from confluent HEK293 WCE with anti-meckelin Ct (‘Ct Ab’) and anti-MKS1 but not by pre-immune serum-negative control or two irrelevant antibodies (Ab1 and Ab2). Right panel: pull-down of filamin A from WCE by GST-tagged meckelin Ct but not by GST alone, or blank and beads only negative (−ve) controls. Western immunoblotting (IB) was with an anti-filamin A MAb. Ten percent of total input WCE is indicated. (E) Co-immunostaining and confocal microscopy of a ciliated mouse IMCD3 epithelial cell for MKS1 (a basal body marker; green in merged image) and filamin A (red) showing localization of filamin A to the basal body and apical cell surface. A 2 μm apical section is shown. Enlarged insets (white frames, labelled a–d) show detail of localization at the basal body (arrows a–d). Scale bar, 5 µm. The asterisk indicates a cell above the monolayer.