Fig. 4. RPGR binds and regulates activity of the actin-severing protein cofilin.

a Mass spectrometry analysis of Rpgr^Ex3d8 retina shows dysregulation of actin regulators (PFN1, PDLIM4, PPP1CC labelled in yellow; two-tailed, unpaired t-test, not corrected for multiple hypothesis testing). b Representative immunoblotting shows cofilin hyperphosphorylation at serine 3 (and therefore reduced activity) (y axis denotes phospho-cofilin:total cofilin ratio; N = 5 control animals, 7 mutant animals; **p = 0.0013; two-tailed, unpaired t-test; error bars denote SEM). c Immunohistochemistry shows cofilin localisation to photoreceptor connecting cilium (CC) in wild-type retinas, partially lost in Rpgr^Ex3d8 mice, not seen in Cofilin knockout mice. White boxes define regions of interest depicted in bottom left panels (blue arrows highlight CC cofilin in wild type retina). Enlarged images (bottom right panels) highlight cofilin colocalization with CC actin in wild type photoreceptors. (Similar results seen in 2 independent experiments using separate animals). d Immunoprecipitation of wild type retinal lysates using magnetic beads coated (+) or uncoated (-) with cofilin antibody shows cofilin enrichment in bound fraction (bottom panel) and RPGR’s retinal specific isoform, detected using GT335 antibody (black arrowhead, top panel; 52 kDa band is acetylated tubulin, bands at 50 kDa and 25 kDa are immunoglobulins). e Transmission electron micrograph of 8-week wild-type photoreceptors show OS composed of compacted discs extending to underlying retinal pigment epithelium (RPE) (left panel). Disc compaction is compromised in age-matched, Cofilin knockout photoreceptors, with discs appearing split and spaced out (right panel; similar results seen in 3 independent experiments). f Examination of basal discs shows an accumulation of shed vesicles (red arrow) in Cofilin knockout mice. (Scale bars; c = 10 µm top panels; 5 µm bottom left panels; 0.5 µm bottom right panels; e = 2 µm; f = 1 µm). Source data provided as Source Data file.