Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus

Abstract

Background: Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.

Results: Bioinformatic analysis reveals that the Evc and Evc2 genes arose through a duplication event early in metazoan evolution and were subsequently lost in arthropods and nematodes. Here we demonstrate that Evc2 is essential for Hh pathway activation in response to the Smo agonist purmorphamine. A yeast two-hybrid screen using Evc as bait identified Evc2 as an Evc binding partner and we confirmed the interaction by immunoprecipitation. We developed anti-Evc2 antibodies and show that Evc2 and Evc co-localize at the basal body and also on primary cilia. In transfected cells, basal body and cilia localization is observed when Evc and Evc2 constructs are co-transfected but not when either construct is transfected individually. We show that Evc and Evc2 are cilia transmembrane proteins, the C-terminus for both being intracellular and Evc2, but not Evc, having an extracellular portion. Furthermore, Evc is absent at the basal body in Evc2 null cells. Using Western blots of cytoplasmic and nuclear protein, we also demonstrate that full length Evc2 but not Evc, is located in the nucleus.

Conclusions: We demonstrate for the first time that Evc2 is a positive regulator of the Hh signalling pathway and that it is located at the basal body of primary cilia. We show that the presence of Evc and Evc2 at the basal body and cilia membrane is co-dependent. In addition, Evc2, but not Evc, is present in the cell nucleus suggesting movement of Evc2 between the cilium and nucleus.

Figure 1

Evc2 is a positive regulator of Hh signalling. (a and b). Representative reporter assays in LIGHT2 Gli-reporter cells (a) and MC3T3 cells (b) transfected with siRNA and treated with control DMSO (white bars) or Hh agonist purmorphamine (black bars). Purmorphamine increases expression of the Gli reporter (FF Luc) with respect to the Renilla control (Ren Luc). Evc2 knockdown was confirmed by western blotting (lower images). Evc2 (approximately 140 kDa) was detected using R1656 and α tubulin (50 kDa) was used as a loading control. Cells with reduced Evc2 protein levels (approximately 80% less than normal) have a significant reduction in Gli reporter expression in response to purmorphamine compared to those transfected with the non-targeting siRNA pool. (c). The purmorphamine induced expression of the Hh responsive gene, Ptch1, is significantly diminished in Evc2 null MEFs (-/-) compared to wild-type controls (+/+) as assessed by RT-PCR.

Figure 2

Protein-protein interaction of Evc and Evc2. (a). Directed yeast two-hybrid assay. For each construct, the portion of Evc and Evc2 coding sequence expressed is depicted. Predicted coiled-coil regions are indicated by black boxes. Negative controls (empty vectors pGBKT7 and pGADT7) and positive controls (p53 and Large-T antigen) were included. + indicates colony growth/interaction, - indicates no colony growth/lack of interaction. (b). FLAG-tagged Evc is immunoprecipitated by Myc-tagged Evc2 but not by the Myc epitope alone. Myc-tagged Evc2 is immunoprecipitated by FLAG-tagged Evc but not by the Flag epitope alone. A non-specific band corresponding to the IgG heavy chain (HC) is indicated.

Figure 3

Subcellular localization of native Evc and Evc2. Each row consists of images from one representative cell. (a). Evc (S43B; red) and Evc2 (R1656; green) colocalize at the base of the primary cilium in an IMCD3 cell. γ- and acetylated tubulin antibodies (both blue) identify the centrioles and the primary cilium respectively. Evc2 has an additional pericentriolar distribution. Scale bar 5 μm. (b). Evc is located along the length of the ciliary axoneme in MC3T3 cells. Evc (S43B; red) colocalizes with the primary cilia marker acetylated tubulin (green). Scale bar 10 μm. (c). Evc2 is concentrated at the base of the primary cilium and only partially colocalizes with Evc in MC3T3 cells. Evc (S43B; red) and Evc2 (R1656; green). Scale bar 10 μm.

Figure 4

Evc and Evc2 localize to the ciliary membrane in co-transfected IMCD3 cells. Each row consists of images from one representative cell. (a). Single transfection and co-transfection of Evc and Evc2-GFP constructs (the protein expressed is given on the left of each image). Expression of either protein alone does not result in localization on cilium. Evc is identified by S43B immunostaining (Evc ab, red), Evc2 by the GFP tag and cilia by acetylated tubulin staining (tubulin; green or red). Co-expression of both proteins results in their expression on the cilium. (cilia from two independent cells are shown). Merged images also show DAPI staining (blue). (b). Immunodetection of Evc and Evc2-GFP co-expressed in cells treated with (+) or without (-) Triton X100 to permeabilize the membranes and allow access to the antibodies. Evc ab (S43B), raised to Evc aa 459 - 999, does not detect Evc in non-permeabilized cells; Evc2 ab 1 (Y20), raised to a Evc2 peptide between aa 50 - 100, detects Evc2 GFP in non-permeabilized cells and Evc2 ab 2 (R1656), raised to Evc2 aa 780 - 1124, does not detect Evc2 in non-permeabilized cells. (c). Schematic representation of Evc and Evc2 on the ciliary membrane as determined from B, the C-termini of both proteins are intracellular. The regions for which we confirmed interaction are indicated by boxes and the predicted coiled-coil regions are shown as small black squares. Regions that are homologous (EVC: 339 - 889, and EVC2: 469 - 1018) are indicated by shaded rectangles. The epitope regions detected by Evc and Evc2 antibodies are indicated. Scale bar throughout 5 μm.

Figure 5

Evc2 is required for the localization of Evc at the base of primary cilia. (a). RT-PCR amplification products from Evc2 null (-/-) and wild-type (+/+) MEFs. As expected, no Evc2 transcript was detected in the Evc2 null MEFs. A significant amount of Evc transcript was amplified in the Evc2 null MEF sample. Hprt transcript was amplified as a control. (b). Western blot analysis of Evc protein in Evc2 null MEFs. The amount of β-actin detected on the same blot was used as a loading control. Evc (approximately 130 kDa) is present in Evc2 null (-/-) MEFs despite having reduced levels (approximately 50%) (c). Representative immunofluorescent staining of Evc in MEF cells. Despite the presence of protein, Evc (red) was not detected at the base of primary cilia in Evc2 null MEFs (-/-). Primary cilia were identified by the presence of acetylated tubulin (green) and nuclei stained with DAPI (blue). Scale bar 10 μm.

Figure 6

Longitudinal and transverse sections of chondrocyte cilia obtained by transmission electron microscopy. The longitudinal section and red lines (left) indicate the approximate position of transverse sections. Transverse sections of cilia are from wild-type (+/+) and Evc mutant (-/-) chondrocytes cells. In both genotypes, disorganized doublets were found in the distal region (red arrow heads) and the champagne glass structures were found in the proximal region (green arrow heads). Nine triplet microtubules and spike structures (blue arrow heads) could be identified in transverse sections of basal body. Scale bar 100 nm.

Figure 7

Evc2 is present in both the cytoplasm and the nucleus of MEF cells. Western blot analysis of total, cytoplasmic and nuclear proteins from wild-type (+/+) and Evc2 (-/-) (a) and Evc (-/-) (b) MEFs. The purity of the cytoplasmic and nuclear protein samples was confirmed using antibodies to α-tubulin (approximately 50 kDa) exclusively cytoplasmic and c-Jun (approximately 43 kDa), exclusively nuclear. Full-length Evc2 (140 kDa) was detected in both the cytoplasmic and nuclear samples. Full-length Evc protein (130 kDa) was only detected in the cytoplasm. Antibodies to both proteins detected several additional bands that are assumed to be non-specific as they are present in the null MEFs.