Figure 3.
RPGR glutamylation at the connecting cilium is mediated by the CID–BD interaction. (A) Ciliary region of a WT mouse retina stained for RPGR (magenta), GT335 (yellow), RP1 (white; upper panel), and rootletin (cyan; lower panel). White boxes in the RPGR and GT335 channels show a magnified single connecting cilium. Smaller white box in the merged channels panel indicates magnified region shown in inset panel on the right. RPGR localizes to the connecting cilium, equivalent to the transition zone in the primary cilium (for photoreceptor diagram see B). GT335 also stains the transition zone and extends distally beyond the connecting cilium in a domain that overlaps with the photoreceptor protein RP1. The ciliary rootlet, stained by the rootletin antibody, extends proximally away from the connecting cilium. RPGR signal fully overlaps with GT335 signal in the connecting cilium (pink, inset), and glutamylated RPGR accounts entirely for the GT335 signal within the connecting cilium (Sun et al., 2016). (B) Schematic diagram of a photoreceptor cell, highlighting the ciliary region, with different domains stained by antibody markers indicated. (C) The ciliary region of an RPGR knockout (KO) mouse retina stained with RPGR (magenta), GT335 (yellow), and rootletin (cyan). Both RPGR and GT335 signals are absent from the connecting cilium, and GT335 stains only a domain distal from the connecting cilium, likely containing glutamylated tubulin. The connecting cilium appears as a gap between the rootletin and GT335 signals (inset). (D) Representative image of the ciliary region of a RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying WT RPGR. The staining pattern largely resembles that of the WT mouse retina, where RPGR and GT335 colocalize to the connecting cilium and appear pink on the merged image (inset). The injected retina shows some background because recombinant RPGR does not cleanly localize to the connecting cilium, as documented previously (Hong et al., 2005). Importantly, only RPGR localized to the connecting cilium is glutamylated, as evidenced by the GT335 signal. (E) Line trace of GT335 (yellow) and RPGR (magenta) signal along the connecting cilium marked with a white line in the inset of the overlay in D. (F) Representative image of matching ciliary region of an RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying the L1302A mutant form of RPGR. The L1302A mutant localizes to the connecting cilium, like WT RPGR, but is not glutamylated as evidenced by the lack of GT335 signal (inset). (G) Line trace as in E. (H) Representative image of matching ciliary region of an RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying the W1294C (human equivalent W1141C) disease mutant form of RPGR. As with L1302A, there is no co-staining of mutant RPGR with GT335 (inset), indicating lack of glutamylation. (I) Line trace as in E. Only WT RPGR injections (E) show correlated GT335 and RPGR signal. (J) Ratio of GT335:RPGR signal at point of maximum RPGR signal obtained from line traces of multiple connecting cilia for WT and RPGR mutants. Each data point indicates an individual cilium analyzed. Bars indicate mean ± SD. P values from two-tailed unpaired t test. n = 30 cilia for WT (three injections), n = 30 cilia for L1302A (three injections), and n = 20 cilia for W1294C (two injections). Data points originating from separate injections are indicated by squares, circles, and triangles. For additional examples of intensity profiles see Fig. S3. Refer to the image caption for details. Panel A shows fluorescence images of R P G R, G T 335, R P 1, and rootletin localization in cilia. Panel B shows a diagram that illustrates photoreceptor cell structure and key ciliary regions. Panel C shows knockout retina images representing the absence of R P G R and reduced G T 335 signals. Panel D shows wild-type R P G R restoration images, which shows colocalization of R P G R and G T 335 signals. Panel E shows an intensity profile graph representing overlapping R P G R and G T 335 signals along the cilium. Panel F shows mutant L 1302 A images with R P G R present but lacking G T 335 colocalization. Panel G shows the intensity profile with reduced overlap between mutant R P G R and G T 335. Panel H shows disease mutant images that show R P G R without a corresponding G T 335 fluorescence signal. Panel I shows an intensity profile that confirms weak overlap between mutant R P G R and G T 335. Panel J shows a scatter plot comparing G T 335 to R P G R signal ratios across conditions.

RPGR glutamylation at the connecting cilium is mediated by the CID–BD interaction. (A) Ciliary region of a WT mouse retina stained for RPGR (magenta), GT335 (yellow), RP1 (white; upper panel), and rootletin (cyan; lower panel). White boxes in the RPGR and GT335 channels show a magnified single connecting cilium. Smaller white box in the merged channels panel indicates magnified region shown in inset panel on the right. RPGR localizes to the connecting cilium, equivalent to the transition zone in the primary cilium (for photoreceptor diagram see B). GT335 also stains the transition zone and extends distally beyond the connecting cilium in a domain that overlaps with the photoreceptor protein RP1. The ciliary rootlet, stained by the rootletin antibody, extends proximally away from the connecting cilium. RPGR signal fully overlaps with GT335 signal in the connecting cilium (pink, inset), and glutamylated RPGR accounts entirely for the GT335 signal within the connecting cilium (Sun et al., 2016). (B) Schematic diagram of a photoreceptor cell, highlighting the ciliary region, with different domains stained by antibody markers indicated. (C) The ciliary region of an RPGR knockout (KO) mouse retina stained with RPGR (magenta), GT335 (yellow), and rootletin (cyan). Both RPGR and GT335 signals are absent from the connecting cilium, and GT335 stains only a domain distal from the connecting cilium, likely containing glutamylated tubulin. The connecting cilium appears as a gap between the rootletin and GT335 signals (inset). (D) Representative image of the ciliary region of a RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying WT RPGR. The staining pattern largely resembles that of the WT mouse retina, where RPGR and GT335 colocalize to the connecting cilium and appear pink on the merged image (inset). The injected retina shows some background because recombinant RPGR does not cleanly localize to the connecting cilium, as documented previously (Hong et al., 2005). Importantly, only RPGR localized to the connecting cilium is glutamylated, as evidenced by the GT335 signal. (E) Line trace of GT335 (yellow) and RPGR (magenta) signal along the connecting cilium marked with a white line in the inset of the overlay in D. (F) Representative image of matching ciliary region of an RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying the L1302A mutant form of RPGR. The L1302A mutant localizes to the connecting cilium, like WT RPGR, but is not glutamylated as evidenced by the lack of GT335 signal (inset). (G) Line trace as in E. (H) Representative image of matching ciliary region of an RPGR knockout (KO) mouse injected subretinally with an AAV vector carrying the W1294C (human equivalent W1141C) disease mutant form of RPGR. As with L1302A, there is no co-staining of mutant RPGR with GT335 (inset), indicating lack of glutamylation. (I) Line trace as in E. Only WT RPGR injections (E) show correlated GT335 and RPGR signal. (J) Ratio of GT335:RPGR signal at point of maximum RPGR signal obtained from line traces of multiple connecting cilia for WT and RPGR mutants. Each data point indicates an individual cilium analyzed. Bars indicate mean ± SD. P values from two-tailed unpaired t test. n = 30 cilia for WT (three injections), n = 30 cilia for L1302A (three injections), and n = 20 cilia for W1294C (two injections). Data points originating from separate injections are indicated by squares, circles, and triangles. For additional examples of intensity profiles see Fig. S3.

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