FIGURE 3:

FHDC1-induced cilia elongation is FH2- and MTBD-dependent. A series of FHDC1 derivatives were expressed in NIH 3T3s by transient transfection. Cilia formation was induced by incubation in low-serum media, the transfected cells were fixed 48 h later ,and the effects on ciliogenesis were assessed by immunofluorescence using an anti-acetylated tubulin antibody (green). FHDC1 expression was detected by virtue of the N-terminal flag epitope tag (red). (A) Expression of full-length FHDC1 inhibits primary cilia formation, but when present the cilia are greatly elongated. (B) FHDC1.I180A-expressing cells assemble primary cilia in most cases. The cilia are longer than control cilia, but do not display the same variation in length induced by FHDC1. (C) Expression of FHDC1.958N had no effect on the number of ciliated cells nor on cilia length. (D) Expression of FHDC1.485N did not affect the number of cells with cilia; however, the cilia were noticeably shorter than on controls. (E) Expression of FHDC1.486C had effects on cilia number and length similar to those of expression of FHDC1.I180A. (F) Schematic of FHDC1 derivatives. Boxes 1 through 5 indicate regions of homology conserved between FHDC1 orthologs. Boxes 4 and 5 comprise the MTBD. (G, H) Quantification of data shown in A–E. N = 3, >100 cells counted per sample. Error bars = SEM. *indicates significant difference from mCherry-expressing controls, p < 0.01.