Shifting distribution of autoradiographic label in cone outer segments and its implications for renewal

To clarify how newly-synthesized membrane materials become distributed within the outer segments (OS) of cone photoreceptors during renewal, the retinas of Xenopus laevis were processed for autoradiography at short times after intraocular injection of radioactive glycoprotein precursors (3H-leucine or fucose). Autoradiograms were examined by light microscopy and the labelling density (number of silver grains divided by surface area) was analyzed as a function of COS height. At the first time (2 hours post-injection) that significant label was observed over COS, it was present at all COS heights but not homogeneously distributed. At this time the labelling density in the most basal compartment was significantly greater than that in the rest of the COS, and in some COS the silver grains seemed to form lines of label parallel to the lamellae. At later times, lines were not observed and the labelling density in the most basal compartment had decreased such that at 17.5 hours post-injection the labelling density in the basal and distal regions of COS was similar. At still later times the labelling density in the most basal compartment was significantly less than that in the rest of the COS. Thus, the initial labelling over COS was not diffuse, and during the first 23 hours after injection the location of highest labelling within COS shifted from the base towards the tip. These observations indicate that new membrane does not quickly diffuse so as to become homogeneously distributed throughout the entire COS, nor does it flow exclusively into new lamellae forming at the COS base. The findings are entirely consistent with, and thereby support, an alternative model, namely that new membrane entering COS flows into partial membrane folds that are known from ultrastructural studies of COS to occur both at the extreme base (basal evaginations) and distributed throughout more distal heights (distal invaginations). The diffuse distribution of label observed over COS at later times is used to derive a revised kinetics for COS renewal, which may have implications for cone sensitivity.