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. 2015 Sep 29;15(2):426–444. doi: 10.1074/mcp.M115.055079

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© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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Fig. 5. — Rapid mTORC1 repression bidirectionally modifies subcellular expression of SNAP-25 and GAP-43. A, Representative images of neurons showing differential expression of SNAP-25 (red) colocalizing with the postsynaptic density protein PSD-95 (green) and the dendritic protein MAP2 (blue) when mTORC1 is active (DMSO, left panel) or repressed (Rapamycin, right panel). Scale bar = 20 μm. See also supplemental Fig. S3. B, Blown up dendrites, indicated by dotted box in A, of SNAP-25 (top), PSD-95 (middle) and merged image showing colocalization (bottom). White arrows indicate SNAP-25 that colocalize with PSD-95. Blue arrows indicate SNAP-25 that colocalize with synapsin. C, Same dendrite as in B, representative staining of the presynaptic protein synapsin (top) colocalized with SNAP-25 (bottom, merged). Scale bar = 5 μm. D, Normalized PCC averaged over many dendrites for SNAP-25 and the postsynaptic marker PSD-95 or the presynaptic marker synapsin and SNAP-25. All raw PCC values were normalized to the average PCC between SNAP-25 and synapsin in control condition (DMSO, D). Dotted line indicates the average normalized PCC between SNAP-25 and synapsin in control condition. Note, there was an increase in the PCC of SNAP-25 with PSD-95 in rapamycin treatment (DMSO, D, = 1.63 ± 0.12, n = 34 dendrites; Rapamycin, R, = 2.12 ± 0.07, n = 24 dendrites). There was no significant difference in PCC between treatments for SNAP-25 and synapsin (DMSO = 1.00 ± 0.13, n = 34 dendrites; Rapamycin = 1.16 ± 0.11, n = 24 dendrites). E, Representative images of neurons showing differential expression of GAP-43 (red), PSD-95 (green), and MAP2 (blue) when mTORC1 is active (DMSO, left panel) or repressed (Rapamycin, right panel). Scale bar 20 μm. F, Blown up dendrites, indicated by dotted box in E, of GAP-43 (top), PSD-95 (middle) and merged image showing colocalization (bottom). White arrows indicate GAP-43 that colocalize with PSD-95. Blue arrows indicate GAP-43 that colocalize with synapsin. See also supplemental Fig. S3. G, Same dendrite as in F, representative staining of synapsin (top) colocalized with GAP-43 (bottom, merged). Scale bar = 5 μm. H, Normalized PCC averaged over many dendrites for GAP-43 and the postsynaptic marker PSD-95 or the presynaptic marker synapsin and GAP-43. All raw PCC values were normalized to the average PCC between GAP-43 and synapsin in control condition (DMSO, D). Dotted line indicates the average normalized PCC between GAP-43 and synapsin in control condition. Note, there was a decrease in the normalized PCC of GAP-43 with PSD-95 in rapamycin treatment (DMSO, D, = 1.36 ± 0.08, n = 36 dendrites; Rapamycin, R, = 1.00 ± 0.09, n = 36 dendrites). There was no significant difference in PCC between treatments for GAP-43 and synapsin (DMSO = 1.00 ± 0.09, n = 36 dendrites; Rapamycin = 0.92 ± 0.08, n = 36 dendrites). Statistics: *, significantly different by one-way ANOVA, Tukey's multiple comparison; ^#, significantly different by single t test from PCC of SNAP-25 or GAP-43 with synapsin in DMSO. Chicken anti-MAP2 was imaged with 405 nm laser (see “Experimental Procedures”).