Abstract
Purpose: :
Kinesin-II is an anterograde molecular motor localized to the connecting cilium of the mammalian photoreceptor. The role of kinesin-II in cone intraflagellar transport (IFT) was examined by cell-specific deletion of Kif3a, the motor subunit of Kinesin-II.
Methods: :
For cone-specific Kif3a deletion, Kif3a flox/flox mice were bred with transgenic mice expressing Cre recombinase under the control of the human red/green pigment (HRGP) promoter. Consequences of Kif3a deletion were analyzed by confocal immunocytochemistry, immunoblotting, ERG and quantitative RT/PCR.
Results: :
Deletion of cone Kif3a resulted in a significant mislocalization of S- and M/L- cone opsins (normally in the outer segments) to the inner segments, outer nuclear layer and synaptic terminals. Rhodopsin kinase (GRK1) and guanylate cyclase (GC1), which are expressed in both rods and cones, were significantly reduced in cone outer segments. Finally, cone-specific phosphodiesterase (cPDE) and transducin were virtually absent from Kif3a-deleted photoreceptors. Real time PCR shows that the decrease is not due to downregulation of mRNA expression and is therefore likely a result of posttranslational degradation. Loss of these proteins essential for phototransduction resulted in complete absence of a light-stimulated response detected by photopic electroretinogram. While GRK1, GC1, cone PDE, and cone transducin are virtually absent from cone outer segments, cone arrestin and the synaptic terminal proteins complexin IV, bassoon and SV2 show normal localizations.
Conclusions: :
Our results suggest that kinesin-II is responsible for intraflagellar transport of the integral membrane proteins S-opsin, M/L-opsin, GC1, as well as membrane-associated phototransduction proteins, to the cone outer segments.
Keywords: photoreceptors • transgenics/knock-outs • color vision