Live-Cell Imaging Evidence for the Ciliary Transport of Rod Photoreceptor Opsin by Heterotrimeric Kinesin-2

Abstract

Primary cilia detect extracellular signals through membrane receptors and channels. The outer segment of a vertebrate photoreceptor cell represents the most elaborate of all primary cilia, containing extraordinarily large amounts of the visual receptor protein, opsin. Because of its high abundance, opsin represents a potential model system for the study of ciliary membrane receptors, including their transport. Here, we have analyzed the movement of ciliary opsin to test whether the highly conserved intraflagellar transport (IFT), as driven by heterotrimeric kinesin-2, is required. Results show that opsin can enter and move along the primary cilium of a nonphotoreceptor cell (an hTERT-RPE1 epithelial cell), suggesting that it can co-opt the basic anterograde motor system of cilia. Fluorescence recovery after photobleaching analysis of cilia of hTERT-RPE1 cells showed that the movement of ciliary opsin was comparable to that of the IFT protein, IFT88. Moreover, the movement of opsin in these cilia, as well as in cilia of mouse rod photoreceptor cells, was reduced significantly when KIF3A, the obligate motor subunit of heterotrimeric kinesin-2, was deficient. These studies therefore provide evidence from live-cell analysis that the conserved heterotrimeric kinesin-2 is required for the normal transport of opsin along the ciliary plasma membrane.