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125I-calmodulin gel overlay techniques have been used to identify calmodulin-binding proteins in teleost retina, in a rod fragment preparation which contains rod inner and outer segments (RIS-ROS), and in RIS-ROS cytoskeletons. We have previously shown that teleost rods change length in response to changes in light conditions, that rod movement is mediated by the actin filaments in the rod inner segment, and that both Ca2+ and cAMP appear to be involved in regulating rod movement. We report here the development of a rod fragment preparation (RIS-ROS), which retains the movable part of the rod, for use in biochemical analysis of rod motility. Gel overlay studies indicate that isolated whole retinas have six prominent calmodulin-binding proteins, migrating at 240 K, 190 K, 150 K, 61 K and a doublet at 18/19 K. In contrast, detached RIS-ROS have three different prominent calmodulin-binding proteins, migrating at 330 K, 33 K, and 31 K. RIS-ROS cytoskeletons have been produced by extraction with Triton X-100; they contain both actin filament bundles and microtubules associated with the connecting cilium. RIS-ROS cytoskeletons have 3 prominent calmodulin-binding proteins migrating at 240 and 18/19 K. These proteins produce faint bands in gel overlays of intact RIS-ROS, but prominent bands in overlays of whole retina. The 240 K protein of RIS-ROS cytoskeletons co-migrates with the 240 K calmodulin-binding subunit of rat brain fodrin. We suggest that the rod 240 K calmodulin-binding protein may be a spectrin-like protein which participates in Ca2+- and calmodulin-regulation of rod motility.

This laboratory has been using the teleost retinal cone as a model for studying the mechanisms and regulation of retinal cell motility. In previous inhibitor studies, the authors have shown that dark-induced cone elongation requires microtubules, whereas light-induced contraction requires actin filaments. This study examines the distributions of actin filaments, microtubules, and intermediate filaments in the cone cytoskeleton. Actin filaments have been localized in isolated cones by labeling with fluorescent derivatives of phalloidin; microtubules were localized by immunofluorescent labeling with anti-tubulin. Actin, microtubule, and intermediate filament distributions have also been examined in detergent-lysed motile cell models of cones fixed with a new method that enhances preservation of the cytoskeleton. Longitudinal bundles of actin filaments extend from the cone's calycal processes through the ellipsoid and into the myoid. No actin filaments are detectable in the perinuclear region and axon, but filaments are present in both pre- and post-synaptic components of the synapse. Intermediate filaments are numerous in the perinuclear region and cone axon but relatively sparse in the myoid. In contrast, microtubule distribution is more uniform: numerous longitudinally oriented microtubules are present throughout the length of the cell. Thus the cone cytoskeleton reflects the highly polarized shape and function of the cell, with actin filaments localized to the distal movable part of the cell and intermediate filaments localized to the proximal part of the cell, which is anchored in the retina.

Nonneural cell extracts contain a heat-stable, nondialyzable activity that will inhibit the spontaneous assembly in vitro of partially purified brain tubulin. The sensitivity of this inhibitory activity to ribonucleases but not to a variety of other hydrolytic enzymes indicates that the inhibitor is an RNA. This conclusion is supported by the observation that purified RNAs from sea urchins, chinese hamster ovary cells, and brain all inhibit spontaneous microtubule assembly in vitro. The synthetic polynucleotides [poly(A), (C), (G), and (U)] are also inhibitory. This inhibition, however, appears to be nonspecific since the RNA base composition is unimportant and a variety of other nonnucleic acid polyanions also function as inhibitors. The treatment of assembly competent tubulin preparations with an insoluble RNA in the form of poly(A) covalently linked to agarose beads produces a "stripped" tubulin which will not assemble microtubules unless a heat-stable, trypsin-sensitive fraction eluted with increased ionic strength is mixed with the "stripped" tubulin. Similar results can be obtained with other cation exchangers, including phosphocellulose and carboxymethylcellulose. The heat-stable protein sequestered by poly(A)-agarose appears to be identical to the "tau" factor recently described by Kirschner and coworkers. Reconstitution experiments indicate that there is a stoichiometric requirement for these factors. These results suggest that spontaneous assembly of microtubules in nonneural cell extracts is blocked because the endogenous factors are complexed with RNA. This idea is supported by the observation that the ratio of tubulin to RNA is low in cultured cell extracts but very high in neural tissue extracts.