Abstract
BackgroundVertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains.ResultsWe examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size.ConclusionsOur observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain.
Highlights
Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment
In wild-type rods at 6 d, Rhodopsin localized only to the outer segment (Fig. 1B, 2B), and we found that Rhodopsin localization is normal in all our transgenic lines (Figs. 1 and 2 and data not shown), indicating that overexpression of either the intracellular or extracellular domains of Crb2a does not interfere with Rhodopsin transport or targeting even though many transgene products localized to the outer segment
In Drosophila photoreceptors, both the extracellular and intracellular domains are important for Drosophila photoreceptor development and morphogenesis, in contrast, the extracellular domain seems largely dispensable for embryogenesis [4,5,15,37]
Summary
Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Vertebrate photoreceptors are morphologically complex and highly compartmentalized cells with large apical domains. The apical domain consists of a proximal inner segment and a distal outer segment, which is a modified cilium packed with intramembranous discs containing the photon-capturing opsins. Little is known about the molecular and cellular mechanisms that control vertebrate photoreceptor morphogenesis and even less about how photoreceptors control the size of their outer segments through the processes of shedding and renewal
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