LOCALIZATION OF KINESIN SUPERFAMILY PROTEINS TO THE CONNECTING CILIUMOF FISH PHOTORECEPTORS

Kinesin superfamily proteins (KIFs) are probable motors in vesicular a nd non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamy domonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only str uctural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel thro ugh which all material passes into and out of the outer segment from t he rod cell body. We have performed immunological tests on isolated su nfish rod inner-outer segments (RIS-ROS) using two antibodies that rec ognize the conserved motor domain of numerous KIFs (anti-LAGSE, a pept ide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at similar to 117 kDa, which is likely to be kinesin heavy chain , and Klp1 head antibody detected a single band at similar to 170 kDa; KIF3A antibody detected a polypeptide at similar to 85 kDa which co-m igrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE an d anti-Klp1 head antibodies detected epitopes in the axoneme and ellip soid, and immunoelectron microscopy with the LAGSE antibody showed tha t the connecting cilium region was particularly antigenic. Immunofluor escence with anti-KIF3A showed prominent labelling of the connecting c ilium and the area surrounding its basal body; the outer segment axone me and parts of the inner segment coincident with microtubules were al so labelled. We propose that these putative kinesin superfamily protei ns may be involved in the translocation of material between the rod in ner and outer segments.