The role of Rab27a in the regulation of melanosome distribution within retinal pigment epithelial cells

Abstract

Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

Figure 1.

Light microscopy reveals normal organization of the retina but altered pigment granule distribution in the RPE of the ashen mouse. Transverse paraffin sections (3 μm) of a wild-type (A) and ashen mouse retina (B) at the age of 5 months stained with hematoxylin and eosin (magnification, approximately ×250). The bottom panels show a higher magnification of RPE from wild-type (C) and ashen mouse (D) (magnification, approximately ×1000). SC, sclera; CH, choroid; RPE, retinal pigment epithelium; ROS, photoreceptor segments of rods and cones; ONL, nuclei of rods and cones; OSL, outer synaptic layer; INL, bipolar cell nuclear layer; ISL, inner synaptic layer; GCL, ganglion cell layer; M, slender glial (Muller) cell processes; G, ganglion cells; IM, inner limiting membrane; N, RPE nucleus; AP, apical processes.

Figure 2.

Electron microscopy shows that melanosomes do not move into the apical processes of RPE of ashen and shaker-1 mouse. Transmission EM longitudinal sections of mouse retinas (A–C) show elongated melanosomes within the apical processes of wild-type RPE that extend between the rod outer segments (ROS), whereas in ashen and shaker-1 mice melanosomes do not enter the apical processes. BM, Bruch's membrane. Oblique sections (D and E) show that many melanosomes are above the level of the adherens junctions (arrows) and extend into the apical processes between the ROS in the heterozygous (+/–, phenotypically normal) mouse (D), whereas in the homozygous (–/–) ashen mouse (E) the melanosomes remain below the level of the junctions. Bar, 2 μm.

Figure 3.

Daily movement of melanosomes within the RPE. 57BL/6J mice were sacrificed just before light onset (A), 1.25 h (B), 2 h (C), 3.5 h (D), and 5.5 h (E) after light onset, and longitudinal sections of RPE were examined by transmission EM. Just before light onset not many melanosomes have entered the apical processes and those that have are in the base of the process. After 1.25 h phagosomes (arrows) are visible in the apical cytoplasm, but few melanosomes are in the apical processes. After 2 h more melanosomes are in the apical processes and some have penetrated into the region between the ROS. Bar, 2 μm.

Figure 4.

Ultrastructure of the apical processes of mouse RPE. Wild-type (A, D, and E), ashen (B and F), and shaker-1 (C and G) RPE were processed for transmission EM (A–D) or scanning EM (E–G). Several very thin apical processes can be seen between adjacent photoreceptor outer segments in wild-type, ashen, and shaker-1. The plasma membrane of the apical process becomes distended and distorted around the pigment granule (D). Scanning EM shows that apical processes are overlapping leaf-like projections in wild-type, ashen, and shaker-1. Bar, 500 nm (A–D); 2 μm (E–G).

Figure 5.

Immunofluorescence localization of melanosomes, Rab27a, and cytoskeletal proteins. Wild-type (A, B, and D–F) and ashen (C) RPE and choroid were labeled with fluorescent phalloidin (red in A–C, E, and F), antitubulin (green in A–C, red in D), anti-Rab27a (green in D), antimyosin VIIa (green in E), and antimyosin Va (green in F). Melanosomes identified by transmitted light have been artificially colored blue in the merged images. Arrows indicate examples of coincidence between melanosomes and rab27a (D) and melanosomes and myosin VIIa (E). AP, apical processes; CB, cell body; BI, basal infoldings. Bar, 5 μm.

Figure 6.

EM localization of melanosomes and F-actin. Cryosections of wild-type RPE were labeled with biotinylated phalloidin, antibiotin antibody and protein A gold (10 nm). Actin staining is just beneath the apical plasma membrane and within the apical processes (A). Melanosomes below the adherens junctions (AJ) are devoid of actin staining (asterisks in B), whereas those above the junctions are closely associated with F-actin staining (arrowheads in B). Oblique sections across the leaf-like processes (C and D) show the distension of the apical processes and the actin cytoskeleton within it that occurs around the pigment granule. Bar, 500 nm.

Figure 7.

EM localization of Rab 27a, myosin VIIa, and myosin Va. Cryosections of wild-type RPE were labeled with 4B12 mouse anti-Rab27a (A and C), and Q142 rabbit anti-Rab27a antibody (B), anti-myosin VIIa (D and E), or antimyosin Va (F and G). Protein A gold particles (10 nm) are indicated in the lower magnification panels by arrowheads. Rab27a is found on the perimeter membrane of most melanosomes, whether elliptical or spherical or in the cell body or the apical processes. Rab27a is also sometimes found within the lumen of the melanosome (B). Myosin VIIa is found on some melanosomes and in the cytoplasm, whereas myosin V is not found on melanosomes but strongly labels the plasma membrane of rod outer segments. Bar, 200 nm.

Figure 8.

Electrophysiological evaluation of retinal function in ashen mice. (A) Representative individual traces of dark-adapted (upper row) and light-adapted (lower row) ERGs in an ashen mouse (left column) in comparison to control mouse (right column). Flash intensities ranged from 10^–4 to 25 cd*s/m². (B) Group comparison of ashen and wild-type mice. Displayed is the dark-adapted (DA) and light-adapted (LA) b-wave amplitude vs. the logarithm of the stimulus intensity. The response distribution characteristics of the ashen mice are shown as box-and-whisker-plots featuring 5 and 95% quantiles (whiskers), 25 and 75% quartiles (box), and the median (marked by an asterisk). The 5 and 95% quantiles of the control mice, indicating the normal range, are given as solid lines, revealing that the responses of the ashen mice were within normal limits.