Abstract
Purpose::
To determine if cellular retinol binding protein (CRBP1) is a plausible candidate for regulating photoreceptor outer segment assembly.
Methods::
After applying 2D-DIGE analysis to cultured intact Xenopus laevis tadpole eyes, we determined that the level of CRBP1 protein was significantly reduced in RPE-deprived retinas with disorganized photoreceptor outer segments compared to RPE-supported eyes with properly folded outer segments. Quantitative Western blots and immunohistochemistry were used to confirm the reduction of CRBP1 levels in RPE-deprived retinas. To determine that the downstream effects of the decreased level of CRBP1 protein were directly responsible for the misfolding of nascent photoreceptor outer segment membranes, we utilized Morpholino antisense oligonucleotides to specifically reduce the translation of CRBP1 protein.
Results::
Quantitative Western blots confirmed that the level of CRBP1 protein was reduced in retinas with disorganized outer segments. Within RPE-supported retinas, CRBP1 protein was present at high levels in both the RPE and Muller cells. In RPE-deprived retinas, the level of CRBP1 was reduced, yet it remained localized within Muller cells. Preliminary results from Morpholino studies indicated that specific reduction of CRBP1 protein levels in RPE-supported retinas resulted in aberrant assembly of photoreceptor outer segment membranes.
Conclusions::
While the literature indicates that CRBP1 is synthesized exclusively by the RPE, we detected the protein within the neural retina of eyes cultured in an RPE-deprived state, thus suggesting that there is likely another source of retinal CRBP1 that has not yet been discovered. Disruption of outer segment assembly in RPE-supported eyes exposed to Morpholinos strongly indicates that CRBP1 is part of a gene/protein network that is critical for the proper folding of outer segments. The localization of CRBP1 within Muller cells further strengthens the potential role of this cell in modulating photoreceptor outer segment assembly.
Keywords: photoreceptors • Muller cells • development