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

Hsu YC, Jensen AM - BMC Cell Biol. (2010)

Bottom Line: We 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT

Background: Vertebrate 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.

Results: We 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.

Conclusions: Our 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.

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Effects of Crb2a constructs that lack the extracellular domain on endogenous Crb2a. (A-E') Confocal z-projections of 6 d rods labeled with zs-4 (blue) and anti-GFP (green) antibodies. (A, A') Wild-type rods. (B, B') Crb2aIntraWT transgenic rods. (C, C') Crb2aIntraΔFBD transgenic rods. (D, D') Crb2aIntraΔPBD transgenic rods. (E, E') Crb2aIntraΔFBDΔPBD transgenic rods. Scale bar, 5 μm.
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Figure 7: Effects of Crb2a constructs that lack the extracellular domain on endogenous Crb2a. (A-E') Confocal z-projections of 6 d rods labeled with zs-4 (blue) and anti-GFP (green) antibodies. (A, A') Wild-type rods. (B, B') Crb2aIntraWT transgenic rods. (C, C') Crb2aIntraΔFBD transgenic rods. (D, D') Crb2aIntraΔPBD transgenic rods. (E, E') Crb2aIntraΔFBDΔPBD transgenic rods. Scale bar, 5 μm.

Mentions: To address whether expression of the intracellular constructs caused mislocalization of endogenous Crb2a, we used zs-4 antibody to localize endogenous Crb2a in Crb2aIntraWT, Crb2aIntraΔFBD, Crb2aIntraΔPBD and Crb2aIntraΔFBDΔPBD transgenic lines (Fig. 7). We found that zs-4 labeling in Crb2aIntraWT (Fig. 7B), Crb2aIntraΔFBD (Fig. 7C), and Crb2aIntraΔPBD (Fig. 7D) and Crb2aIntraΔFBDΔnΔPBD (Fig. 7E) was similar to that in wildtypes (Fig. 7A), indicating that overexpression of these constructs did not alter endogenous Crb2a localization.


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

Hsu YC, Jensen AM - BMC Cell Biol. (2010)

Effects of Crb2a constructs that lack the extracellular domain on endogenous Crb2a. (A-E') Confocal z-projections of 6 d rods labeled with zs-4 (blue) and anti-GFP (green) antibodies. (A, A') Wild-type rods. (B, B') Crb2aIntraWT transgenic rods. (C, C') Crb2aIntraΔFBD transgenic rods. (D, D') Crb2aIntraΔPBD transgenic rods. (E, E') Crb2aIntraΔFBDΔPBD transgenic rods. Scale bar, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927502&req=5

Figure 7: Effects of Crb2a constructs that lack the extracellular domain on endogenous Crb2a. (A-E') Confocal z-projections of 6 d rods labeled with zs-4 (blue) and anti-GFP (green) antibodies. (A, A') Wild-type rods. (B, B') Crb2aIntraWT transgenic rods. (C, C') Crb2aIntraΔFBD transgenic rods. (D, D') Crb2aIntraΔPBD transgenic rods. (E, E') Crb2aIntraΔFBDΔPBD transgenic rods. Scale bar, 5 μm.
Mentions: To address whether expression of the intracellular constructs caused mislocalization of endogenous Crb2a, we used zs-4 antibody to localize endogenous Crb2a in Crb2aIntraWT, Crb2aIntraΔFBD, Crb2aIntraΔPBD and Crb2aIntraΔFBDΔPBD transgenic lines (Fig. 7). We found that zs-4 labeling in Crb2aIntraWT (Fig. 7B), Crb2aIntraΔFBD (Fig. 7C), and Crb2aIntraΔPBD (Fig. 7D) and Crb2aIntraΔFBDΔnΔPBD (Fig. 7E) was similar to that in wildtypes (Fig. 7A), indicating that overexpression of these constructs did not alter endogenous Crb2a localization.

Bottom Line: We 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Massachusetts, Amherst, MA 01003, USA.

ABSTRACT

Background: Vertebrate 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.

Results: We 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.

Conclusions: Our 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.

Show MeSH
Related in: MedlinePlus