Fig 1.
EYS has four isoforms annotated in genomic and protein databases: a summary of the genomic and protein structure of EYS isoforms.
(A) A schematic view of EYS transcript variants. Perpendicular bars represent protein coding exons, horizontal lines represent introns and arrows indicate the transcriptional direction (data from NCBI Gene database; http://www.ncbi.nlm.nih.gov/gene/346007). (B) Predicted protein domain structure of EYS isoforms; domain order of EYS isoforms 1 and 4 is presented as previously described whereas the domain structure of EYS isoforms 2 and 3 was predicated using the Prosite ExPasy database.
Table 1.
An overview of human EYS isoforms.
Fig 2.
EYS isoforms are expressed in the retina, testis and Y79 retinoblastoma cell line: RT-PCR analysis of mRNA expression of EYS isoforms in human tissues and cell lines.
(A) mRNA transcripts of EYS isoforms 2 and 3 are present in the cDNA from the human retina, testis and Y79 cell line. A fragment of the HPRT gene was used as a quantitative control and a reaction lacking template cDNA was used as a negative control (-ve control). (B) EYS isoform 4 is expressed in Y79 cell line as confirmed by amplification of EYS isoform 4-specific exon (upper band). In the positive control (+ve control), pcDNA3.1-EYS isoform 1 plasmid was used as template whereas a reaction lacking template cDNA was used as a negative control (-ve control).
Fig 3.
EYS localises to the cell cytoplasm, centrosomes and ciliary axoneme: immunofluorescent localisation and Western blot analysis in Y79 cells.
(A) Immunofluorescent localisation of EYS (green) in a cluster of Y79 cells. (B) Co-labelling of EYS (green) with Acetylated-α-tubulin (Ac- α-tubulin; red). (C) Co-labelling of EYS (green) and F-actin (red) in dbcAMP treated Y79 cells. (D) Co-labelling of EYS (green) and CEP135 (red). (E) High magnification showing co-labelling of EYS (green) with Ac-α-tubulin (red) in a ciliated cell. The zoomed inserts represent the areas demarcated by the dashed boxes; the yellow arrows indicate the cilium. Cell nuclei are stained with DAPI (blue). Scale bars: 10 μm. (F) Western blot analysis of the presence of EYS in total protein extract versus membrane protein fraction obtained from Y79 cells. β-dystroglycan (β-DG) was used as an internal control and loading was optimised by BCA assay – 40 μg of protein were loaded in each well.
Fig 4.
eGFP tagged fragments of EYS isoform 1 localise to the cell cytoplasm: immunocytochemical analysis in HeLa cells.
(A) Localisation of the eGFP tagged N-terminal fragment of EYS isoforms 1 (1-1635 aa; eGFP-EYS N-term) in cells stained with WGA (red). (B) Localisation of the eGFP tagged C-terminal fragment (1880–3144 aa; eGFP-EYS C-term) in cells stained with WGA (red). (C) Localisation of eGFP tag in cells stained with WGA–eGFP tag-only control. The zoomed inserts represent the areas demarcated by the dashed boxes. Cell nuclei are stained with DAPI (blue). Scale bars: 10 μm.
Fig 5.
eGFP tagged EYS isoforms 2 and 3 localise to the cell cytoplasm: immunocytochemical analysis in Y79 cells.
eGFP empty vector was used as a control and Texas Red-X conjugated WGA was used to stain the cell membrane. The zoomed inserts represent the areas demarcated by the dashed boxes. Cell nuclei are stained with DAPI (blue). Scale bars: 10 μm.
Fig 6.
eGFP tagged EYS isoforms 2 and 3 localise to the cell cytoplasm and may form dimers: immunocytochemical and Western blot analysis in HeLa cells.
(A) Localisation of the eGFP tagged EYS isoforms 2 and 3 in cells stained with WGA (red). (B) Localisation of the eGFP tagged EYS isoforms 2 and 3 in cells stained with phalloidin (red). eGFP empty vector was used as a control. The zoomed inserts represent the areas demarcated by the dashed boxes. Cell nuclei are stained with DAPI (blue). Scale bars: 10 μm. (C) Western blot analysis of FLAG tagged EYS isoforms 2 and 3. Protein extract from untransfected HeLa cells was used as a negative control. Loading was optimised by BCA assay – 40 μg of protein were loaded to each well.
Fig 7.
Fluorescently labelled EYS is detected as a whip-like signal in the outer retina: immunohistochemical analysis in macaque retinal cryosections.
(A) Fluorescent localisation of EYS (green) in macaque retinal cryosections. The zoomed images represent the areas demarcated by dashed boxes. (B) The negative control (-ve control) was obtained by omitting the primary antibody. (C) The DIC (differential interference contrast) image illustrates the cytoarchitecture of the analysed photoreceptor cells. The layers of the retina are labelled as follows: RPE—retinal pigment epithelium, OS–outer segment, CC–connecting cilum, IS–inner segment, ONL–outer nuclear layer, OPL–outer plexiform layer, INL–inner nuclear later, IPL–inner plexiform layer, GCL–ganglion cell layer. Cell nuclei are labelled with DAPI (blue). Scale bars: 20 μm.
Fig 8.
Fluorescently labelled EYS overlaps with markers of the outer segment and the ciliary axoneme: immunohistochemical analysis in macaque retinal cryosections.
(A) EYS (green) overlaps with Arrestin (red) in the photoreceptor outer segments, the white arrows indicate an exemplary photoreceptor outer segment. (B) EYS (green) partially overlaps with Acetylated α-tubulin (Ac-α-tubulin; red). (C) A high magnification image demonstrating the overlap of EYS (green) and Ac-α-tubulin (red) in a single cell, the white arrows indicate the ciliary axoneme. (D) EYS (green) partially overlaps with RP1 (red). (E) A high magnification image demonstrating the overlap of EYS (green) and RP1 (red) in a single cell, the white arrows indicate the ciliary axoneme. The layers of the retina are labelled as follows: RPE—retinal pigment epithelium, OS–outer segment, IS–inner segment. Cell nuclei are labelled with DAPI (blue). Scale bars: 20 μm.
Fig 9.
EYS is expressed in rods and cones: immunohistochemical analysis in macaque retinal cryosections.
(A) EYS (green) is concentrated on one side of the rod outer segments labelled with Rhodopsin (red). The white arrows indicate the analysed rod outer segment. (B) EYS (green) concentrates unilaterally in the cone outer segment highlighted by PNA staining (red). The white arrows indicate the analysed cone outer segment. (C) EYS (green) concentrates unilaterally in the cone outer segment highlighted by S-opsin (red). The white arrows indicate the analysed cone outer segment. The layers of the retina are labelled as follows: OS–outer segment, IS–inner segment. Scale bars: 20 μm.