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Fig. 1. The Caenorhabditis elegans CaMKII ortholog UNC-43 is localized to neurites. (A,B) UNC-43::CFP and (C,D) GLR-1::YFP can be detected in perinuclear structures in neuronal cell bodies (A,C) and clusters within the neurites of the ventral nerve cord (B,D). Merged images for A-D indicate that UNC-43 and GLR-1 colocalize (arrowheads) at perinuclear structures (E), and at a fraction of neurite clusters (F). Arrow in E indicates a unipolar neurite, partially out of the focal plane, extending from the cell body. Gray-scale images of UNC-43::GFP (G) and UNC-43(T286D)::GFP (H) indicate that the T286D change increases the amount of UNC-43 found diffusely distributed throughout the neurites. (I) Real-time PCR measurements of unc-43::gfp mRNA for nematodes expressing either one or two copies of an unc-43::gfp integrated transgene. Values are normalized to real-time PCR measurements of a dlg-1 control transcript. A significant difference in the level of expression was observed, ^**P<0.001 using Student's t-test (n=4), between the two groups. Fluorescence quantification measuring the mean fluorescent intensity values for neuron cell bodies (J) or neurites expressing either one copy or two copies of an unc-43::gfp transgene integrated into the genome (K). Fluorescence quantification measuring the mean fluorescent intensity values for neuron cell bodies (L), neurites including clustered and unlocalized fluorescence (M) and unlocalized fluorescence alone (N) (i.e., diffuse fluorescence defined by a threshold within two standard deviation values of the mean outside of the clustered signal) for nematodes expressing either UNC-43::GFP or UNC-43(T286D)::GFP. (O) The number of clusters (clusters defined as above a threshold of two standard deviation values of the mean outside of the clustered signal) per 10 µm length of neurite. (P) Frequency distribution plots of the mean fluorescence values of UNC-43::GFP clusters (straight black line) or UNC-43(T286D)::GFP clusters (dashed red line). A.U., arbitrary fluorescence units. Error bars indicate s.e.m. Significant differences ^*P<0.01, ^***P<0.0001 were observed using the Student's t-test between the indicated test groups and the WT or single copy control group. n=10-25 animals per genotype. Bar 5 µm (A-F); 10 µm (G-H).

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