To determine the distribution of rhodopsin and other outer segment proteins within rod photoreceptors, frozen retinal sections of S334ter
+, rho
+/− mice were labeled with the anti-rhodopsin antibody, Rho4D2, and anti-rom-1, Rom C-1 acidic. Rho4D2 antibody binds to the N terminus of rhodopsin and thus recognizes both endogenous and mutant forms. Full-length rhodopsin was localized to the outer segments of rho
+/− photoreceptors, and was weakly detected in the ONL
(Fig. 2A) . Rhodopsin was detected in various membrane domains within the CTCWTKO (S334ter
+, rho
+/−) photoreceptors including the outer segment, inner segment, perinuclear region, and synaptic terminal
(Fig. 2B) . Immunohistochemistry performed on CTCKO (S334ter
+, rho
−/−) mice photoreceptors lacking endogenous rhodopsin also showed prominent mislocalization of truncated rhodopsin within the inner segment, cell body, and synaptic terminal membranes
(Fig. 2C) . Although long outer segments do not form in CTCKO animals, membranes distal to the inner segment were labeled with rhodopsin antibody
(Fig. 2C) . The disc rim protein, Rom-1, was localized to outer segments in both WTKO and CTCWTKO mice, indicating that the presence of truncated rhodopsin does not alter the localization of Rom-1 (data not shown). In CTCKO photoreceptors, Rom-1 trafficked to the distal connecting cilium, was incorporated into ciliary membranes, and was virtually absent from inner segments and the cell body (data not shown). This observation is similar to Rom-1 localization in rhodopsin-knockout mice,
25 and suggests that the mechanisms governing outer segment protein transport are not severely altered in CTCKO mice, despite the absence of large, organized outer segments.