The Role of Rab27a in the Regulation of the Distribution of Melanosomes within the RPE
To determine whether Rab27a regulates melanosome distribution in the retina, we examined the distribution of melanosomes within the RPE of homozygous ashen (Rab27a null) mice, heterozygous, and wild-type mice 2 h after light onset. Histological sections observed under a light microscope revealed that the gross organization of the retina is normal. All retinal layers are present with normal thickness but the melanosome distribution within the ashen RPE is different from the wild type (). Melanosomes in wild-type RPE are found in an area that extends clearly apical to the nucleus, whereas the distribution of melanosomes within ashen RPE is much more restricted in that melanosomes are apparently absent from the area apical to the nucleus ().
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 (more ...)
To confirm these findings, we subjected mouse retinas to thin-section electron microscopy (EM). EM analysis showed that in wild-type mice many melanosomes of the RPE are distributed within the cell body but some are found within the apical processes of the RPE interdigitating with the outer segments of photoreceptors (). In contrast, the melanosomes in the ashen retina remain in the cell body and are completely absent from the apical processes (). A considerable proportion of the melanosomes in the RPE of wild-type mice are elliptical in shape and oriented with their longer axis parallel to the longitudinal axis of the cell. This orientation is evident throughout the RPE cell, both above and below the adherens junctions. The melanosomes of the ashen mouse are more randomly oriented. Examination of multiple longitudinal sections of wild-type and ashen RPE indicates fewer elongated and more spherical melanosomes in ashen RPE. This is likely to be due to the random orientation of the melanosomes such that in thin-section more appear spherical, rather than because of a difference in melanosome shape in the ashen RPE. As not all apical processes of wild-type RPE contain melanosomes, changes in the distribution of melanosomes within the apical processes are most obvious in oblique sections where multiple apical processes and photoreceptor outer segments are within the same section (each RPE cells is exposed to ~40 outer segments). In these sections, it is clear that the melanosomes in homozygous ashen retinas do not pass beyond the level of the adherens junctions (), whereas in heterozygous retinas many melanosomes are seen above the level of the adherens junctions and between photoreceptor outer segments ().
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 (more ...)
The melanosome distribution within the ashen
RPE mimics that of the shaker-1
RPE (; Liu et al., 1998
) as they are also completely absent from the apical processes and show a more random orientation.
Daily Movement of Melanosomes within the RPE
Ever since the first microscopic studies of retinas over a century ago, it has been suggested that melanosome movement in mammals is minimal and very little is known about melanosome dynamics within mammalian RPE cells. The above results suggested that RPE melanosomes indeed move and that the movement may be regulated by at least two gene products, Rab27a and Myosin VIIa. To study RPE melanosome dynamics, we determined whether melanosomes of the RPE change their distribution with the daily light cycle by examining the distribution of RPE melanosomes of wild-type mice sacrificed at different times. Immediately before light onset, we observed only a small number of melanosomes in the apical processes and those are, in general, at or near the base below the outer segments (). Just >1 h after light onset, we observed a large number of phagosomes present in the apical cytoplasm. These phagosomes are predictably the product of light-induced phagocytosis of photoreceptor outer segments (). At this time point, the number of melanosomes in the apical processes remains low but the number increases significantly 1 h later. Although many of these melanosomes are still in the base of the apical processes at the 2 h after light onset time point, a significant number of melanosomes extend into the interdigitating processes (). At later time points (3.5 and 5.5 h after light onset), there is a gradual reduction in the number of melanosomes in the apical processes (). Quantitation of the number of melanosomes in the apical processes at different time points indicates that there is a maximum of a threefold increase in the number of melanosomes in the apical processes 2 h after light onset, compared with the dark-adapted eye ().
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 (more ...)
Quantitation of the number of melanosomes within the apical processes at different times in the light cycle
Ultrastructure of the Apical Processes of Mouse RPE
Transmission EM of the wild-type mouse RPE suggested there is very little space between adjacent outer segments within which the melanosomes must move. This prompted us to examine the melanosomes within the apical processes at high magnification in order to further understand the relationship between the melanosome, the cytoskeleton, and the plasma membrane of the apical process. High magnification shows multiple very thin apical processes extending between the outer segments in the wild-type, ashen, and shaker-1 RPE (). These images suggested that the apical processes of the mutant ashen and shaker-1 RPE might be more disorganized than those of the wild-type RPE, but this is difficult to resolve by thin-section EM, where an entire apical process may not be within the section plane. It is also not possible to assess the three-dimensional organization of the apical processes by thin-section EM. We therefore performed scanning EM of the mouse RPE after removal of the photoreceptor layer. The apical processes of RPE cells are not finger-like projections but rather overlapping leaf-like projections (). Those projections in the ashen and shaker-1 RPE show no clear differences from the wild type. Although the melanosomes that enter the wild-type apical processes are elongated, their shortest diameter is still greater than the width of the apical processes when they do not contain melanosomes. When a melanosome enters an apical process, the membrane of the apical process appears to become distended around the granule (). It is not possible even at the highest magnification to visualize cytoskeletal structures within the apical processes whether or not they contain melanosomes.
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 (more ...)
Interaction of Melanosomes of RPE with Cytoskeleton
Fluorescent phalloidin staining of wild-type RPE shows F-actin to be localized to the basal infoldings and to the apical processes (). F-actin is also found in the circumferential actin ring in the apical region of the cell, which is associated with the adherens junctions. In these 0.5-μm sections that do not contain the entire cell, the circumferential actin ring appears as a short stretch of filament bundle only (). The apical processes are not rigid because after paraformaldehyde fixation the photoreceptors become partially or totally separated from the RPE. The RPE–photoreceptor interface is thus not as well preserved as in the conventional EM shown above, where glutaraldehyde fixation was used. Microtubules are confined to the cell body and absent from the apical processes. Most melanosomes are found in the microtubule-rich cell body but some have penetrated the actin-rich region of the cell, moving beyond the actin-rich cortex and into the actin-rich apical processes. Both melanosomes and actin are concentrated in the basal region of the apical processes and appear closely associated. In the ashen RPE, the organization of actin filaments and microtubules appears the same as the wild-type but the melanosomes are all within the microtubule-rich cell body and show no association with F-actin ().
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, (more ...)
Rab27a is associated with the melanosomes within the wild-type RPE, particularly with the elongated melanosomes in the apical region of the cell but also with some spherical melanosomes in the cell body (). Myosin VIIa stains at least some of the melanosomes in the RPE by immunofluorescence and also shows some cytoplasmic staining (). Some bright staining is also observed above the cell body of the RPE, which may be on outer segments of photoreceptors that have remained associated with the RPE (). Myosin Va shows a distribution that is very different from that of myosin VIIa. Myosin Va strongly stains structures that are clearly apical to the region containing the majority of the F-actin and melanosomes (). These structures are photoreceptor outer segments (see below), which sometimes remain associated with RPE after paraformaldehyde fixation.
Cryo-immuno-EM allowed the identification of the adherens junctions and revealed F-actin labeling in the infoldings of the basal membrane (unpublished observations) and in the circumferential actin ring, just beneath the apical plasma membrane, and within the apical processes (). Melanosomes that were at the level of or above the adherens junctions had F-actin closely associated with them, whereas those below the adherens junctions were completely devoid of actin staining. Rab27a was found on all types of melanosomes within the RPE, whether present in the cell body or in the actin-rich apical regions and whether spherical or elliptical in shape (). Although the majority of the Rab27a staining was around the perimeter membrane of the melanosome, some Rab27a staining was observed within the lumen of the granule. Myosin VIIa staining was found on some melanosomes within the RPE. The staining was observed on both spherical and elliptical melanosomes in the cell body, both above and below the adherens junctions and within the apical processes. Nearly all the staining was present around the perimeter membrane of the melanosome (). Myosin Va localized to the plasma membrane of photoreceptor outer segments and both the apical processes, and melanosomes were largely devoid of myosin Va staining ().
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 (more ...)
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 (more ...)
Electroretinography Analysis of Ashen Mice
The potential impact of the lack of Rab27a on retinal function was assessed with electroretinography (ERG). Under scotopic conditions with low stimulus intensities (up to ~10–2 cds/m2), the rod system response can be determined, whereas responses of the cone system can be identified under photopic conditions. The scotopic ERG with stimulus intensities of 10–1 cds/m2 and more represents a mixed response of rods and cones. summarizes the results obtained in ashen (Rab27a null) mice in comparison to wild-type controls.
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 (more ...)
A series of exemplary scotopic and photopic ERG traces obtained from an ashen and a wild-type control mouse is shown in . No significant differences in the shape of the waveforms or the size of the responses could be identified. A comparison between both groups regarding the photopic and scotopic b-wave amplitude is shown in . The upper and lower lines delimit the normal range (95 and 5% quantiles of the control group), whereas the ashen data are shown as box plots (the gray boxes indicate 25 and 75% quantiles, the whiskers the 5 and 95% quantiles). The results demonstrate that the b-wave amplitude of the ashen mice was within normal limits for all stimulus intensities under both photopic and scotopic conditions. In summary, no functional differences between ashen and wild-type mice were found.