Evidence That the Crumbs Complex Is a Key Component of the Outer Segment Renewal Mechanism

Abstract

Purpose:: The cellular and molecular mechanisms that control the fascinating and essential process of photoreceptor outer segment renewal have remained unidentified, although it is clear from studies made decades ago that outer segment renewal is regulated by light and the light-cycle. Recently we showed that the FERM protein Moe directly interacts with Crumbs proteins and moe^- rods have significantly larger outer segments than normal (Development 133:4849-59, 2006). Based on our observations, we proposed that Moe is a negative regulator Crumbs protein function in photoreceptors and that the Crumbs complex is a good candidate to be part of the mechanism that controls outer segment renewal. We now have more compelling data that supports these ideas.

Methods:: We have made stable zebrafish transgenic lines where different HA-tagged constructs of the transmembrane bound intracellular domain of zebrafish (crb2a^Intra) are expressed in rod photoreceptors.

Results:: We found that expression of crb2a^IntraDD (two residues in the FERM binding domain predicted to be phosphorylated were mutated to Asp (D) to act as phosphomimetics) causes a striking phenotype; stripes of Crb2a^IntraDD protein are observed in outer segments and the number of stripes correlates with the number of light cycles the cells have experienced. Further, outer segment morphology is strikingly similar to moe^- rods. Our preliminary observations show that when zebrafish larvae are placed in constant dark for many days, we no longer observe stripes of Crb2a^IntraDD protein; this fits perfectly with the observations made decades ago that constant dark slows or stops outer segment renewal. In comparison, we found that Crb2a^IntraWT protein levels are much less than Crb2a^IntraDD levels, suggesting that the phosphorylation state of Crb2a^Intra is an important regulator of Crumbs protein levels. Weak stripes of Crb2a^IntraWT are still observed in outer segments that correlate with the number of light cycles. We are further characterizing the morphology of Crb2a^IntraWT and Crb2a^IntraDD rods and analyzing effects of other Crumbs constructs on rod morphology and outer segment renewal.

Conclusions:: Our observations offer a compelling case that Crumbs proteins (and their regulators) are a key component of the outer segment renewal mechanism which would make it the first such component to be identified. We propose that stimulation of Crumbs activity may be an ideal target for preserving photoreceptor function (and survival) by maintaining normal outer segment length in photoreceptor degeneration diseases that are marked by progressive outer segment shortening.

Keywords: protein structure/function • neuroprotection • regeneration