Characterization of peripherin/rds and rom-1 transport in rod photoreceptors of transgenic and knockout animals.
OBJECTIVE
Peripherin/rds and rom-1 have structural roles in morphogenesis and stabilization of the outer segment, but little is known about their transport and sorting to the rod outer segment. Peripherin/rds and rom-1 trafficking were studied in several knockout and transgenic animal models.


METHODS
Rod outer segment formation and distribution of peripherin/rds and rom-1 were examined by immunohistochemistry, electron microscopy, and molecular biological methods in wild-type, rhodopsin-knockout, and peripherin/rds-knockout mice. C-terminally truncated peripherin/rds (Xper38)-GFP chimeric protein sorting was followed by immunofluorescence microscopy in transgenic Xenopus.


RESULTS
In developing wild-type photoreceptors, peripherin/rds was detected exclusively in the distal tip of the connecting cilium in advance of outer segment formation. Rhodopsin-knockout mice failed to create normal rod outer segments and instead, elaborated membranous protrusions at the distal cilium tip. Peripherin/rds and rom-1 localized to this ciliary membrane in rhodopsinless photoreceptors. In transgenic Xenopus, a C-terminally truncated peripherin/rds-GFP fusion predominantly localized to its normal location within disc rims. In developing rds mice, rom-1 accumulated primarily in distal ciliary membranes.


CONCLUSIONS
Peripherin/rds transport and localization are polarized to the site of outer segment morphogenesis before disc formation in developing photoreceptors. Peripherin/rds and rom-1 trafficking is maintained in rhodopsin-knockouts, suggesting that rim proteins and rhodopsin have separate transport pathways. The presence of truncated peripherin/rds-GFP in the outer segment supports previous evidence that peripherin/rds mice form homotetramers for outer segment targeting. The finding that rom-1 transports to the outer segment domain in rds mice suggests that rom-1 may possess its own sorting and transport signals.
Lee
Edwin S
ES
Department of Molecular Cell Biology, University of California, Berkeley, USA.
Burnside
Beth
B
Flannery
John G
JG
D000818:Animals; D030801:Animals, Genetically Modified; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008855:Microscopy, Electron, Scanning; D016253:Microscopy, Immunoelectron; D009024:Morphogenesis; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D064531:Peripherins; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics; D012374:Rod Cell Outer Segment / Q000378:metabolism* / Q000648:ultrastructure; D059470:Tetraspanins; D014158:Transcription, Genetic; D029867:Xenopus Proteins; D014982:Xenopus laevis
D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C578995:PRPH protein, Xenopus; D064531:Peripherins; C578996:Prph2 protein, mouse; C579003:Prph2 protein, rat; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C081477:Rom1 protein, mouse; D059470:Tetraspanins; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D012243:Rhodopsin