Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

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.

The Par-PrkC Polarity Complex Is Required for Cilia Growth in Zebrafish Photoreceptors

“…The loss of crb2b function in zebrafish photoreceptors reduced apical membrane size similar to our results with Pard3 and PrkC [28]. In contrast, overexpression of various domains of crb2a significantly increased the size of the apical membrane and outer segment [29]. Whether these protein complexes function together or in a sequential process remains poorly understood.…”

“…Loss of the zebrafish ortholog crb2b results in a reduction of apical domain size and loss of outer segments [28], while mutation of Crumbs homolog 1 (CRB1) causes early vision loss and photoreceptor degeneration [32], [33]. Overexpression of the intracellular domain of Crb2a in zebrafish photoreceptors leads to an expansion of the outer segments, supporting a role for the Crb complex in positively regulating the size of the apical domain, including cilia [29]. The role other apical determinants play in ciliogenesis remains less clear.…”

Unique cell biological profiles of retinal disease-causing missense mutations in the polarity protein Crumbs

“…Loss of mouse Crb1 function causes a disorganization of the retinal layers and the formation of rosette-like structures, defects indicative of a loss of integrity in the retinal epithelium (Mehalow et al, 2003;van de Pavert et al, 2004). Similar defects were seen in zebrafish embryos that lacked the function of Crb2a (Omori and Malicki, 2006;Hsu and Jensen, 2010). Gross morphological defects are preceded by problems in the integrity of the zonula adherens (ZA; the outer limiting membrane of the vertebrate retina) that emerge during terminal differentiation of the retina, and a failure to form inner and/or outer segments of normal size.…”

Characterization of the cell polarity gene crumbs during the early development and maintenance of the squid–vibrio light organ symbiosis

“…As a complementary experiment, we examined histological samples of the light organ and eye using paraffin-section ISH methods as previously described (Peyer et al 2014). In the eye, we localized es-crumbs along with es-rhodopsin to confirm that both genes occur in regions of the photoreceptors similar to other animals (Kingston et al 2015; Hsu and Jensen 2010). We included rhodopsin because it is a common reference gene in the eye.…”