Abstract
Purpose::
Caveolin-1 (Cav-1) is a lipid raft-associated protein expressed in several retinal cell types, but its function in photoreceptors has not been determined. We have observed reduced retinal function in Cav-1 global null mice but this phenotype is complicated by the expression of Cav-1 in a variety of retinal cell-types. To gain insight into Cav-1 function in photoreceptors specifically, we have generated rod photoreceptor-specific transgenic Xenopus laevis that express Cav-1 fused to enhanced green fluorescent protein (eGFP) and the fusion protein product has been localized by confocal and fluorescence microscopy.
Methods::
The retinally-expressed isoform of Xenopus laevis Cav-1 (BC070672) was amplified from Xenopus retinal cDNA and cloned into an expression plasmid contain eGFP in frame fusion with the Cav-1 insert downstream of the 1.3 kb Xenopus opsin promoter. Transgenic frogs were generated by by random insertion of the linearized transgene construct into frog sperm DNA and injection into frog oocytes. Transgenic tadpoles were screened by detection of intrinsic eGFP fluorescence emitted from pupils. Localization of the transgene product was determined by both fluorescence and confocal microscopy and eGFP-Cav-1 localization was compared with several lipid raft, photoreceptor, and cytoskeletal proteins. Confirmation of transgene expression was also determined by immunoblot analysis.
Results::
Expressed eGFP-Cav-1 fusion proteins were associated with rod photoreceptor plasma membranes of the inner segment, cell body, and synapses. In addition, eGFP-Cav-1 was clearly associated with intracellular vesicular structures in the inner segment. The fusion protein was enriched at a boundary zone between the outer and inner segments. Most dramatically, eGFP-Cav-1 localized to discrete projections to the outer segment that were aligned with filamentous actin as visualized by rhodamine-conjugated phalloidin.
Conclusions::
The localization to vesicular structures and to the outer/inner segment boundary suggests a possible role in intracellar trafficking of material to or from the outer segment. The association of eGFP-Cav-1 with filamentous actin in discrete outer segment projections suggests localization to either calycal processes or disk incisures. Future experiments will test a role in intracellular trafficking using rod-specific expression of dominant-negative Cav-1 mutants.
Keywords: photoreceptors • transgenics/knock-outs • lipids