Figure 5. Calcyon regulates trafficking of ZnT3 to mossy fibers.

A. Sedimentation profile of ZnT3 in glycerol gradient fractions prepared from wild type (WT) and Cal^−/− brains determined by immunoblotting with ZnT3 antibodies. B. Distribution of ZnT3 across the WT (closed circles) and Cal^−/− (open circles) gradients following normalization to the total present in the WT gradients. Positions of molecular weight markers are shown to the left. C, D. ZnT3 immunostaining of hippocampal cryosections from WT (C) and Cal^−/− (D) mice at low (left) and high (right) magnification. E. Fluorescent staining intensities were obtained in circles (shown in yellow) of equal area positioned over the hilus. Values of ZnT3 staining in Cal^−/− samples were normalized to those detected in WT samples in the same rostral-caudal position. Scatter plot shows values for each sample and horizontal line, the SEM. Compared to levels detected in WT, ZnT3 levels in the hilus of the dentate gyrus are significantly reduced in Cal^−/− brain (**, p<0.01, t-test). F. Higher magnification (40×) view of ZnT3 staining (green) in mossy fiber terminals in CA3 region (left), and axons in the hilus of the dentate gyrus (right). Nuclei were detected with DAPI (blue). The axon to terminal staining ratio for each sample was determined following measurement of ZnT3 labeling in the hilus and CA3 area using circles of equal size as described above. Bar= 50 µM. G. Histogram with bars and error bars showing the mean CA3/Hilus ratio, and the SEM for the WT and Cal^−/− samples. The ratio is significantly reduced in Cal^−/− samples suggesting impaired sorting of ZnT3 to terminals in the CA3 (*, p<0.05 two-tailed paired t-test).