Our aim was to use native and genetically modified mice in conjunction with microscopy, HPLC, and immunocytochemistry to understand mechanisms governing the retinoid cycle and visual transduction. Spectrally sensitive noninvasive two-photon microscopy in conjunction with genetically engineered mice lacking key components of the retinoid cycle was used to identify a novel structure in the RPE involved in the production of the visual chromophore, 11-cis-retinal. Two-photon microscopy permitted deep tissue penetration of infrared excitation light in anesthetized mice to monitor regeneration processes of rhodopsin without introducing external fluorophores. Our results revealed the existence of novel structures, RESTs (or retinosomes), that are critical to the formation of 11-cis-retinal.
In wild-type mice, all-trans-retinol exchanges rapidly between the blood and the RPE (Qtaishat et al., 2003
). RESTs appear to be essential structures for retaining esterified vitamin A in the eye to support its utilization in formation of the chromophore for visual pigments. RESTs are also essential for trapping all-trans-retinol generated in photoreceptors, which must diffuse across the ECM separating ROSs and the RPE. Subcellular localization of these structures allows efficient trapping of all-trans-retinol from both the choroidal circulation and from photoreceptors after photoisomerization of rhodopsin's chromophore. This process is stalled in the RPE of Rpe65−/−
mice (Redmond et al., 1998
; Qtaishat et al., 2003
) that overaccumulate all-trans-retinyl esters in aberrantly large RESTs ().
The flow of soluble retinoids in either free or protein-bound form is governed by the gradient generated by the conversion of 11-cis- and all-trans-retinol to insoluble all-trans-retinyl esters in the RPE and the conversion of 11-cis-retinal to 11-cis-retinylidene-opsin in rod photoreceptor cells (McBee et al., 2001
). Hence, compartmentalization plays an essential role in driving energetically unfavorable chemical reactions through mass action (McBee et al., 2001
). The all-trans-retinyl esters constitute a storage intermediate in the isomerization pathway from all-trans- to 11-cis-isomers (Rando, 1996
). The isomerization proceeds through a reaction that involves an unidentified enzyme-retinol intermediate, or a specific subpopulation of all-trans-retinyl esters with properties that are distinct from the bulk of all-trans-retinyl esters (Stecher et al., 1999
). All-trans-retinyl esters present in the RESTs and formed in situ on the internal membranes where LRAT resides may form two subpopulations, from which only one is used for the isomerization process directly.
Several molecular components involved in the 11-cis-retinal formation have been identified through biochemical and genetic approaches. RPE65 is thought to be involved in the delivery of all-trans-retinyl esters to the isomerization machinery by the virtue of specific binding of these esters (Gollapalli et al., 2003
; Mata et al., 2004
). Our analyses add a cell biological view for the formation of retinyl esters during the retinoid cycle. All-trans-retinol likely diffuses first to the ER, where the major fraction is converted to retinyl esters by the ER-localized LRAT. Our in vivo analysis demonstrates that the retinyl esters are next delivered to and stored in RESTs. Blocking the redistribution of all-trans-retinyl esters from RESTs resulted in aberrant accumulation of all-trans-retinyl esters in RESTs as observed in the RPE of Rpe65−/−
mice. In photoreceptors of Lrat−/−
mice regenerated with 9-cis-retinal, photoisomerization of isorhodopsin (9-cis-retinylidene-opsin) produced all-trans-retinoids, which could not be converted to 11-cis-retinal, hence formation of rhodopsin (11-cis-retinylidene-opsin) was not observed. Free all-trans-retinol could be quickly lost to the circulation, whereas all-trans-retinyl esters are essential in the retention of retinoids in the eye. This observation does not discard the possibility that all-trans-retinol is used directly by a putative isomerase, simply because all-trans-retinyl esters could be the starting substrate for the RPE65–hydrolase–isomerase complex indispensable in the production of 11-cis-retinol.
On average, between 20 and 40 ROSs project toward one RPE cell (for review see McBee et al., 2001
). For efficient transfer of retinoid between the RPE and the photoreceptor cells, the retinoid-processing enzymes should be widely distributed throughout the cell as observed in this and previous reports (McBee et al., 2001
; Haeseleer et al., 2002
; Batten et al., 2003
). Once the retinoids are esterified, they are trapped into RESTs (), which are composed of all-trans-retinyl esters and at least one additional protein component, ADRP. The formation of self-associating complexes of all-trans-retinyl esters (Li et al., 1996
) could facilitate REST formation. Clustering of all-trans-retinyl esters may prevent diffusion of retinoids through the retina, and may circumvent overproduction of all-trans-retinoic acid, an agent known to cause cell differentiation and proliferation (Mangelsdorf et al., 1995
), thus lowering overall toxicity. However, the symmetric nature of these structures and their intracellular distribution suggest that perhaps additional proteins are also involved. Interestingly, Liu et al. (2003)
provided evidence that the lipid droplets in CHO K2 cells are metabolic organelles involved in membrane traffic, and that they contained ADRP as a major protein component of these structures termed adiposomes. It is tempting to speculate that ADRP could be an essential component of the lipid structures throughout the body.
RESTs in the RPE. The RPE (hexagons) interdigitate with ROSs. Large curved arrows symbolize the flow of retinoids from and to the rod outer segments (OS). RESTs are depicted as elongated red ovals ~6.9 μm in length.
Prior reports of myeloid bodies in the RPE have demonstrated that they vary in size during light adaptation (Tabor and Fisher, 1983
; Abran and Dickson, 1992a
; Dickson and Morrison, 1993
; Cai and Dickson, 1994
), suggesting possible involvement in the retinoid cycle. However, the appearance and location of the REST is different from the myeloid bodies, and the relationship between these two structures is unclear. Robison and Kuwabara (1977)
observed accumulation of lipid inclusion droplets along the basal and lateral cell boundaries after injection of the substantial dose of all-trans-retinyl esters. In our experiments, the subcellular localization of the RESTs, a minor component of the RPE, and appearance of these structures differ from those previous observations. Most EM analyses were performed on radial sections of the eye, and hence it was difficult to provide quantitative results for the thin structures perpendicularly aligned to the RPE cell layer. A combination of EM and application of two-photon microscopy to study visual processes using the infrared laser for the excitation of the fluorophores opens a new way to detect and study the subcellular structures.
In summary, in this work we have used the power of noninvasive, spectrally sensitive two-photon microscopy in conjunction with genetically engineered mice lacking key components of the retinoid cycle to define a novel structure in the RPE, the RESTs (). Two-photon microscopy has unsurpassed potential to advance our understanding of normal physiological processes and to provide new insights into the pathology of many other eye diseases using suitable mouse models.