The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking
Fig 7
NINL and CC2D2A co-localize with MICAL3 and are required for correct MICAL3 localization.
(a) Schematic of a photoreceptor for orientation. (b-d’) Co-localization of endogenous MICAL3 (green signal; b) and polyglutamylated tubulin (red signal; c) in rat retina (P20) by co-immunostaining radial cryo-sections. The yellow signal in the merged image (d’) indicates co-localization at the base of the photoreceptor connecting cilium. (b’-d’) are high magnification images of the boxed areas in (b-d). (e-f”) Centrosomal co-localization of NINLisoB, CC2D2A and MICAL3 in hTERT-RPE1 cells. mRFP-NINLisoB (red signal, e) localizes to the basal body of the cilia marked with polyglutamylated tubulin (cyanid signal; e) and overlaps with GFP-tagged MICAL3 (green signal, e’) at the ciliary base when co-expressed (yellow signal, e”). Co-expression of eCFP-CC2D2A (green signal, f) and mRFP-MICAL3 (red signal, f’) resulted in partial overlap at the base of the cilia (yellow signal, f”). (g) Endogenous MICAL3 (green signal) detected by immunostaining clusters at the ciliary base (white arrows; cilium marked with anti-acetylated tubulin in red) of hTERT-RPE1 cells treated with non-targeting siRNA. (h, i) Knockdown of NINL (h) or CC2D2A (i) expression by siRNA results in dispersed distribution of MICAL3 throughout the cell body (brackets) with retention of some MICAL3 puncta at the ciliary base (arrows). qPCR analysis of NINL (j) and CC2D2A (k) siRNA treated hTERT-RPE1 cells. Cells were transfected with 10nM siRNA and all qPCR data were normalized against GUSB levels. Bar and error bars refer to mean and standard deviation, respectively (n = 3, on two biologicial replicates). *: P<0.05; **: P<0.01 versus non targeting siRNA (NT) (student’s t-test). Nuclei are counter stained with DAPI in all panels (blue signal). Scale bars: are 5 μm in d, 1 μm in d’ and 10 μm in e-i.