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Mentions: Since both Evc and Evc2 are co-dependent for cilia localization of expressed proteins, we tested if Evc2 is required for cilia localization of Evc in Evc2 cells. MEFs derived from Evc2 mutant mice do not produce any Evc2 transcript or protein (Figure 5a and 6a). Evc and Evc2 are both detected at the base of primary cilia in wild-type MEFs (Figure 5c and not shown, respectively). Despite the presence of Evc transcript and protein (Figure 5a-b) Evc was not detected at the base of cilia in Evc2 MEFs. This confirms that Evc2 is essential for Evc cilia localization.
Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus
Bottom Line: 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.
Affiliation: Institute of Human Genetics, Newcastle University, Centre for Life, Central Parkway, Newcastle Upon Tyne, NE1 3BZ, UK.
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 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.
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