Cell type-specific and light-dependent expression of Rab1 and Rab6 GTPases in mammalian retinas

Abstract

The Ras-like Rab1 and Rab6 GTPases modulate protein traffic along the early secretory pathway and are involved in the regulation of maturation of rhodopsin in the outer retina. However, Rab GTPases have not been studied in the inner retinas. Here, we analyzed the anatomatic distribution and expression of Rab1 and Rab6 in the mouse and rat retinas by immunohistochemistry and immunoblotting. We found that Rab1 was specifically expressed in the rod bipolar cells, while Rab6 was expressed in a different cell type(s) from rod bipolar cells in the inner retina. We also demonstrated that expression of Rab1 and Rab6 was increased with light. These data provided the first evidence implicating that Rab1 and Rab6 may be involved in the regulation of the retinal adaptation.

Fig. 1

Expression of Rab1 (A–E) and Rab6 (F–J) in the transverse sections of the mouse retinas. (A and F) Representative immunostaining with Rab1 (A) and Rab6 (F) antibodies. (B and G) Immunostaining of Rab1 (B) and Rab6 (G) antibodies (red) and nuclear staining of DAPI (blue). (C and H) Enlarged sections of immunostains from the INL to the IPL with Rab1 (C) and Rab6 (H) antibodies. (D and I) Enlarged sections of immunostains from the outer border of ONL with Rab1 (D) and Rab6 (I) antibodies. (E and J) Enlarged sections of immunostains from the GCL with Rab1 (E) and Rab6 (J) antibodies. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar is 50 µm in (A, B, F, and G), and it is 25 µm in (C–E) and (H–J).

Fig. 2

Rab1 (A–D) and Rab6 (E–H) expression in the transverse sections of the rat retinas. (A and E) Immunostaining with Rab1 (A) and Rab6 (E) antibodies. (B and F) Immunostaining of Rab1 (B) and Rab6 (F) antibodies (red) plus DAPI staining of the nuclei (blue). (C and G) Enlarged sections of immunostains from the outer border of ONL with Rab1 (C) and Rab6 (G) antibodies. (D and H) Enlarged sections of immunostains from the GCL with Rab1 (D) and Rab6 (H) antibodies. Scale bar is 50 µm in (A–B) and (E–F); it is 25 µm in (C–D) and (G–H).

Fig. 3

Immunostains of Rab1 (A–C) and Rab6 (D–F) with PKC in the inner mouse retinas. The transverse sections taken from mouse retinas were double stained with antibodies against Rab1 (A–C) or Rab6 (D–F) with PKC, a rod bipolar cell marker. Rab1 colocalized with PKC, while Rab6 did not. Red, Rab staining; green, PKC staining; yellow, colocalization of Rab with PKC. Scale bar: 50 µm.

Fig. 4

Enlarged sections of immunostains of Rab1 (A–C) and Rab6 (D–F) with PKC in the GCL in mouse retinas. The transverse sections taken from mouse retinas were double stained with antibodies against Rab1 (A–C) or Rab6 (D–F) with PKC. Red, Rab staining; green, PKC staining; yellow, colocalization of Rab with PKC; blue, nuclear staining of DAPI. Scale bar is 25 µm in (A–C) and 30 µm in (D–F).

Fig. 5

The transverse sections taken from mouse retinas were double stained with antibodies against Rab1 (A–E) or Rab6 (F–J) with GS, a Müller cell marker. Enlarged sections of immunostains of Rab1 (D–E) and Rab6 (I–J) with GS at the outer border of ONL (D and I) and at the GCL (E and J) in mouse retinas. Neither Rab1 nor Rab6 was colocalized with GS. Red, Rab staining; green, GS staining; blue, nuclear staining of DAPI. Scale bar is 50 µm in (A–C) and (F–H); it is 25 µm in (D–E) and (I–J).

Fig. 6

Rab1 expression in the mouse retinas was enhanced under the light adaptation compared to that under dark adaptation. (A) Comparison of immunostaining with Rab1 antibodies in the light-adapted (left panel) and dark-adapted (right panel) retinas obtained under the identical procedures. From each immunostain, three ROIs, ROI1 (green box), ROI2 (blue box), and ROI3 (brown box), were selected in the outer border of the ONL, the INL, and the innermost of the IPL, respectively, to quantify the intensity of the immunolabeling with Rab1 antibodies. Scale bar: 50 µm. (B) The mean immunofluorescent intensities from the light- and dark-adapted retinas. For each ROI, 20 scan planes were taken at different depths. The mean fluorescent intensities from each scan plane were plotted as a function of its related depth. (C) The average of the mean immunofluorescent intensities of the 20 scan planes from the light- and dark-adapted retinas as shown in (B). *P < 0.05 versus light adaptation.

Fig. 7

Rab6 expression in the mouse retinas was also enhanced under the light adaptation compared to that under dark adaptation. The detailed experimental procedures are essentially the same as described in the legend of Fig. 5 for Rab1. (A) Immunostains of Rab6 antibodies in the light-adapted (left panel) and dark-adapted (right panel) retinas. Scale bar: 50 µm. (B) The mean immunofluorescent intensities of the three ROIs from each scan plane were plotted as a function of its related depth. (C) The average of the mean immunofluorescent intensities of the 20 scan planes from the light- and dark-adapted retinas as shown in (B). *P < 0.05 versus light adaptation.

Fig. 8

Western blot analysis of Rab1 and Rab6 expression in the light- and dark-adapted mouse retinas. (A) Representative blots showing that expression of Rab1 (left panel) and Rab6 (right panel) is increased in the light-adapted retinas (light) as compared to the dark-adapted retinas (dark). The numbers indicated on the left are molecular weights. After immunoblotting with anti-Rab antibodies, the blots were stripped and reprobed with anti-actin antibodies (lower panels), which serve as controls for equal protein loading. (B) Quantitative data expressed as fold increase over the dark-adapted retinas. Similar results were obtained in three independent experiments.