Identification of novel molecular components of the photoreceptor connecting cilium by immunoscreens

The connecting cilium of photoreceptor cells is the only intracellular link between the morphologically, functionally and biochemically different compartments of the inner and outer segments. The non-motile modified cilium plays an important role in the organization and the function of photoreceptor cells, namely in delivery and turnover of enzymes and substrates of the visual transduction cascade, and the photosensitive membranes of the outer segment. The protein components of the cilium participate in the intracellular transport through the cilium, in the outer segment disk morphogenesis and in the maintenance of discrete membrane domains. In order to identify yet unknown cytoskeletal components of the connecting cilium, a combined biochemical and molecular biological strategy was applied. For this purpose, antibodies were raised against proteins of photoreceptor cell axonemes. Using this AX-4-antiserum, a rat retina cDNA expression library was immunoscreened and clones encoding partial sequences of (i) already known photoreceptor specific proteins; (ii) ubiquitously expressed proteins; (iii) clones with homologies to retinal ESTs; and (iv) clones coding for cytoskeletal proteins were isolated. Further analysis revealed that these candidate clones have homologies to Drosophila flightless I, mouse APC-binding protein EB2, human microtubule associated protein 4 (MAP4), human centrin 3, human cytoplasmic dynein intermediate chain 2C, and human dynamitin.The immunoscreening approach used here was successfully applied to isolate genes encoding yet unknown cytoskeletal proteins of photoreceptor cell axonemes. The obtained information will provide further insight into the role of the connecting cilium in photoreceptor cell function.