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. 2020 Mar 4;98(6):1213–1231. doi: 10.1002/jnr.24608

FIGURE 4.

FIGURE 4

Tomosyn knockdown results in increased RhoA activity in hippocampal neurons. Hippocampal neurons were co‐transfected with a RhoA biosensor and either scrambled shRNA or shTomosyn. (a) Representative images show the neurons in the intensity‐modulated display mode. Higher magnification of the soma and a dendritic segment in dashed box areas are enlarged for better visualization. Scale bar, 20 and 5 µm. (b) FRET efficiency was determined as a ratio of YFP/CFP. ****p < 0.0001, unpaired Student's t‐test. n = 72 scramble and 69 shTomosyn. (c) Schematic structure of domain mutant tomosyn. FL‐Tom, full‐length mouse tomosyn (1‐1166 aa); Tom‐ΔC, containing an N‐terminal domain with WD40 repeats (1‐1048 aa); Tom‐ΔN, containing a SNARE coil‐coiled domain (1049‐1109 aa). (d) Western blot shows the expression of domain mutant tomosyn (Tom‐ΔC‐RFP and Tom‐ΔN‐RFP), FL‐Tom‐RFP, and RFP control in N2a cells. (e) Summary graph showing FRET efficiency in domain mutant tomosyn‐expressing neurons. Neurons with Tom‐ΔN‐RFP showed increased FRET efficiency. ***p < 0.001 compared to RFP, FL‐tomosyn, and ΔC‐tomosyn, one‐way ANOVA with Dunnett's multiple comparisons test. n = 32 RFP, 28 FL‐Tom‐RFP, 29 Tom‐ΔC‐RFP, and 32 Tom‐ΔN‐RFP