Analysis of 14-3-3 isoforms expressed in photoreceptors

Abstract

The 14-3-3 family of proteins has undergone considerable expansion in higher eukaryotes with humans and mice expressing seven isoforms (β, ε, η, γ, θ, ζ, and σ) from seven distinct genes (YWHAB, YWAHE, YWHAH, YWHAG, YWHAQ, YWHAZ, and SFN). Growing evidence indicates that while highly conserved, these isoforms are not entirely functionally redundant as they exhibit unique tissue expression profiles, subcellular localization, and biochemical functions. A key limitation in our understanding of 14-3-3 biology lies in our limited knowledge of cell-type specific 14-3-3 expression. Here we provide a characterization of 14-3-3 expression in whole retina and isolated rod photoreceptors using reverse-transcriptase digital droplet PCR. We find that all 14-3-3 genes with the exception of SFN are expressed in mouse retina with YWHAQ and YWHAE being the most highly expressed. Rod photoreceptors are enriched in YWHAE (14-3-3 ε). Immunohistochemistry revealed that 14-3-3 ε and 14-3-3 ζ exhibit unique distributions in photoreceptors with 14-3-3 ε restricted to the inner segment and 14-3-3 ζ localized to the outer segment. Our data demonstrates that, in the retina, 14-3-3 isoforms likely serve specific functions as they exhibit unique expression levels and cell-type specificity. As such, future investigations into 14-3-3 function in rod photoreceptors should be centered on 14-3-3 ε and 14-3-3 ζ, depending on the subcellular region of question.

Keywords: 14-3-3; Cone; Digital droplet PCR; Photoreceptor; Retina; YWHAE; YWHAZ; rd1.

Figure 1

14-3-3 proteins are expressed throughout the retina. A–B) mouse retina labeled with WGA (left), pan 14-3-3 (center, green), with merged images including Hoechst labeling for nuclei on the right. A polyclonal pan 14-3-3 antibody (Abcam) was used in A, and a monoclonal pan-14-3-3 (H8) in B. Asterisks in (B) indicate labeling of blood vessels by the secondary antibody. Scale bar, 20 μm and abbreviations are: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GC, ganglion cell layer. C) Western blots of recombinant 14-3-3 proteins probed with the antibodies used in A–B; blots stained with REVERT to label total protein. Quantitation of the band intensities, data represent mean + SD, reveals no statistically significant differences between the two antibodies.

Figure 2

Six of the seven 14-3-3 isoforms are expressed in retina. A) RT-ddPCR results from mouse retina plotted as a function of starting cDNA concentration. Note, error bars are smaller than the symbol size. B) Human retina RNAseq data replotted from (Whitmore et al., 2014). RT-ddPCR results from rd1 (C) or conefull (D) mouse retina. E) Comparison of the slope of the linear regression for each gene from A, C, and D. P-values are represented as n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Figure 3

YWHAE and YWHAZ are expressed in rods. A) Cell fragments released from retina after papain treatment stained with Hoechst, and in the right panel overlaid with the transmitted light channel. Asterisks indicate rod perikarya; MC is a fragment of a Muller glia cell. B) Profile of sorted cells, area circled in blue containing the cells marked green is the population of rod perikarya collected for RNA extraction. C) RT-ddPCR results from rod perikarya.

Figure 4

14-3-3 ε and ζ are differentially localized in photoreceptors. A) Western blots of recombinant 14-3-3 proteins probed with 14-3-3 ε (upper) or 14-3-3 ζ (lower) antibodies; blots stained with REVERT to label total protein. Mouse retinas labeled with WGA and either 14-3-3 ε (C) or 14-3-3 ζ (D); scale and abbreviations as in Figure 1.

Figure 5

Correlation between phylogeny and retina expression of 14-3-3. Cladogram of 14-3-3 isoforms adjacent to a summary of the findings regarding mRNA and protein expression in the retina, n.c. indicates no change, information corresponding to the blank rows in the protein columns was not determined.