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SA Baker, G Pazour, D Cole, G Witman, JC Besharse; Do Intraflagellar Transport Proteins Assemble into a Complex in the Vertebrate Retina? . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3742.
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Purpose: Intraflagellar Transport (IFT) has been identified in motile flagella of Chlamydomonas and sensory cilia of C. elegans as a mechanism required for assembly and maintenance of cilia and flagella. In IFT, movement of a large complex of ∼17 IFT proteins along axonemal microtubules is catalyzed by Kinesin II and cytoplasmic dynein. Our goal is to use biochemical and immunological techniques to study IFT protein interactions in the mammalian retina. Methods: Indirect immunocytochemistry was performed on fresh frozen bovine retinal sections. Bovine retinal extracts were prepared by homogenization in HMEK buffer. Ciliary axonemes were prepared by detergent extraction of bovine RIS/ROS. Extracts were fractionated on sucrose density gradients and analyzed by western blotting with antibodies raised against the mouse sequences of IFT20, IFT52, IFT57, and IFT88. Results: Immunocytochemistry reveals that each of the four IFT proteins is found throughout the inner segment and concentrated at the basal body-connecting cilum. When retinal extracts are analyzed on sucrose density gradients we find that the IFT proteins are broadly distributed from 21S-3S, suggesting a large monomeric pool of IFT proteins as well as larger protein complexes. An additional pool of IFT proteins tightly associates with the ciliary axoneme and requires harsh chaotropic salts or ionic detergents for extraction. Conclusion: Our data suggest that IFT20, 52, 57, and 88 interact as part of a protein complex as predicted by the IFT model. However, a significant portion of IFT protein in retina exists in both a monomeric pool and in a pool that is bound in a stable fashion to the photoreceptor cytoskeleton.
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