In a recent study, we reported that in bovine brain extract, glycogen synthase kinase-3␤ and tau are parts of an ϳ400 -500 kDa microtubule-associated tau phosphorylation complex (Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002) J. Biol. Chem. 277, 11933-11940). In this study, we find that when purified brain microtubules are subjected to Superose 12 gel filtration column chromatography, the dimeric scaffold protein 14-3-3 coelutes with the tau phosphorylation complex components tau and GSK3␤. From gel filtration fractions containing the tau phosphorylation complex, 14-3-3, GSK3␤, and tau co-immunoprecipitate with each other. From extracts of bovine brain, COS-7 cells, and HEK-293 cells transfected with GSK3␤, 14-3-3 co-precipitates with GSK3␤, indicating that GSK3␤ binds to 14-3-3. From HEK-293 cells transfected with tau, GSK3␤, and 14-3-3 in different combinations, tau co-immunoprecipitates with GSK3␤ only in the presence of 14-3-3. In vitro, ϳ10-fold more tau binds to GSK3␤ in the presence of than in the absence of 14-3-3. In transfected HEK-293 cells, 14-3-3 stimulates GSK3␤-catalyzed tau phosphorylation in a dose-dependent manner. These data indicate that in brain, the 14-3-3 dimer simultaneously binds and bridges tau and GSK3␤ and stimulates GSK3␤-catalyzed tau phosphorylation.

In Alzheimer's disease, microtubule-associated protein tau is hyperphosphorylated by an unknown mechanism and is aggregated into paired helical filaments. Hyperphosphorylation causes loss of tau function, microtubule instability, and neurodegeneration. Glycogen synthase kinase-3␤ (GSK3␤) has been implicated in the phosphorylation of tau in normal and Alzheimer's disease brain. The molecular mechanism of GSK3␤-tau interaction has not been clarified. In this study, we find that when microtubules are disassembled, microtubuleassociated GSK3␤ dissociates from microtubules. From a gel filtration column, the dissociated GSK3␤ elutes as an ϳ400-kDa complex. When fractions containing the ϳ400-kDa complex are chromatographed through an anti-GSK3␤ immunoaffinity column, tau co-elutes with GSK3␤. From fractions containing the ϳ400-kDa complex, both tau and GSK3␤ co-immunoprecipitate with each other. GSK3␤ binds to nonphosphorylated tau, and the GSK3␤-binding region is located within the N-terminal projection domain of tau. In vitro, GSK3␤ associates with microtubules only in the presence of tau. From brain extract, ϳ6-fold more GSK3␤ co-immunoprecipitates with tau than GSK3␣. These data indicate that, in brain, GSK3␤ is bound to tau within a ϳ400-kDa microtubule-associated complex, and GSK3␤ associates with microtubules via tau.

The evolutionarily conserved Semaphorin family proteins are well known axon guidance ligands that mediate both attractive and repulsive responses in invertebrates and vertebrates. In this study, we show that the Drosophila Semaphorin-1a (Sema1a), a transmembrane Semaphorin, is required cell autonomously in adult photoreceptor (R-cell) axons for the establishment of an appropriate topographic termination pattern in the optic lobe. Loss of sema1a disrupts the association of neighboring R-cell growth cones leading to defects in local termination pattern, whereas overexpression of sema1a induces the hyper-fasciculation of R-cell axons. The function of Sema1a in R-cell axon guidance absolutely requires its cytoplasmic domain. We propose that Sema1a functions as a receptor in regulating R-cell axon guidance in the Drosophila visual system.

The nervous system in many species consists of multiple neuronal cell layers, each forming specific connections with neurons in other layers or other regions of the brain. How layer-specific connectivity is established during development remains largely unknown. In the Drosophila adult visual system, photoreceptor (R cell) axons innervate one of two optic ganglia layers; R1-R6 axons connect to the lamina layer, while R7 and R8 axons project through the lamina into the deeper medulla layer. Here, we show that the receptor tyrosine kinase Off-track (Otk) is specifically required for lamina-specific targeting of R1-R6 axons. Otk is highly expressed on R1-R6 growth cones. In the absence of otk, many R1-R6 axons connect abnormally to medulla instead of innervating lamina. We propose that Otk is a receptor or a component of a receptor complex that recognizes a target-derived signal for R1-R6 axons to innervate the lamina layer.