IMMUNOCYTOCHEMICAL LOCALIZATION OF OPSIN IN ROD PHOTORECEPTORS DURINGPERIODS OF RAPID DISC ASSEMBLY

Transport of opsin from photoreceptor inner to outer segments has been assumed to occur via the connecting cilium, the only permanent struct ural connection between these two regions. However, in prior work, lit tle or no immunoreactive opsin has been detected in the cilium, despit e the high rate of transport of this protein. This suggests that immun e epitopes are masked during passage through the cilium or that opsin is transported via an extra-ciliary route. In this study, we stained t he photoreceptors of Xenopus laevis with well-characterized monoclonal antibodies directed at the N-terminal, C-terminal, and 5-6 loop regio ns of bovine opsin. This was done on isolated retinas incubated in vit ro under conditions that support rapid disc assembly, to insure that o psin transport to forming discs was occurring at the time of fixation. Five MAbs that gave robust staining of Xenopus rod inner segment/rod outer segment preparations with the light microscope were utilized for electron microscopic studies on LR White embedded or cryo-ultrathin s ections. Four of these stained outer segment discs and inner segment v esicles and plasma membrane. However, no significant staining of the c onnecting cilium was found. Furthermore, freeze-fractured mouse photor eceptors prepared by the 'fracture-label' technique showed extensive l abelling of membrane compartments but lacked staining of the connectin g cilium. Isolated retinas incubated under conditions that support rob ust rod disc synthesis contained many finger-like and vesicular projec tions of the apical inner segment plasma membrane and inner segment ve sicles extending into them. Rod outer segment nascent discs usually ma de close contact with the inner segment. Both the vesicular profiles a ssociated with the inner segment plasma membrane and the basal discs e xtending to the inner segment were heavily stained with all four anti- opsin antibodies. This suggests an alternate route for bulk transport of opsin to newly forming discs that involves direct transfer from api cal inner segment plasma membrane to nascent discs.