Figure 8.
CaMKIγ and TRPC5 colocalize with lipid raft markers in rat brain and cultured hippocampal neurons. A, Endogenous CaMKK, CaMKIγ, and TRPC5 are present in the lipid raft fraction (fractions 4) as confirmed by enrichment of the raft marker Flotillin-1 in fraction 4 of a discontinuous sucrose density gradient (fractions 1–4, 5% sucrose; fractions 5–11, 35% sucrose; fraction 12, 50% sucrose) from P0 rat forebrains. The input is the crude homogenate before any centrifugation steps separating cytosol and membranes. B, Immunofluorescent staining of endogenous TRPC5 (pseudocolored red) and endogenous Flotillin-1 (pseudocolored green) in axonal growth cone of E18 hippocampal neuron. Scale bar: 5 μm. C, Colocalization of Alexa Fluor 546-CTxB labeled raft marker GM1 (as described in Materials and Methods) and EGFP-CaMKIγ in DIV 1 hippocampal neurons. Scale bar: D, 20 μm. Immunofluorescent staining of endogenous β-tubulin, TRPC5 with electroporated EGFP-CaMKIγ in stage 2 (top row) and stage 3 (bottom row) neurons shows extensive colocalization of TRPC5 and EGFP-CaMKIγ. In final column, the images are a merge of all three channels, with β-tubulin pseudocolored blue, EGFP-CaMKIγ pseudocolored green, and endogenous TRPC5 pseudocolored red in stage 2 and stage 3 hippocampal neurons. The insets show additional magnification of largest growth cones. Scale bars: β-tubulin (bottom) panel, 10 μm; EGFP-CaMKIγ (bottom inset showing growth cone), 5 μm. E, Quantification of neurons with Tau-1-positive axons expressing wild-type CaMKIγ, CaMKIγDPP (mutant lacking prenylation and palmitoylation sites), or CaMKIα fixed at 24 h to evaluate acceleration of axonogenesis. F, Quantification of Tau-1-positive axons in neurons expressing wild-type CaMKIγ alone or wild-type CaMKIγ plus siTRPC5 at 24 h. Note, in D and E, neurons are fixed at 24 h to test mutant CaMKIγ and siTRPC5 in acceleration of axon formation assay. G, Quantification of rescue experiment from neurons expressing control, siTRPC5, or siTRPC5 plus rescue with caCaMKIγ.