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. 2017 Jan 2;216(1):231–246. doi: 10.1083/jcb.201601098

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© 2017 Bruckner et al.

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Figure 1. — Fife interacts with RIM. (A) Fife and RIM domain structures, indicating the interacting Fife C2A and C2B (green) and RIM C2B (red) domains. (B) FLAG-tagged Fife C2A (green, lanes 1–4) and C2B (green, lanes 5–8) specifically precipitate HA-tagged RIM C2B (red). Input lanes contain lysate equal to 10% of the amount used for the pull-down assays. IP indicates the antibody used for immunoprecipitation. IgG bands at 25 kD are not depicted. (C–F) Representative traces of EJPs and mEJPs recorded in 0.6 mM Ca²⁺ at wild type (C), Fife^AC/+ (D), RIM^ex103/+ (E), and Fife^AC+/+RIM^ex103 (F) NMJs. Stimulus artifacts have been removed for clarity. (G) Mean EJP amplitude is significantly reduced in Fife^AC/Fife^AC, Fife^AC/Df, RIM^ex103/RIM^ex73, and Fife^AC+/+RIM^ex103, but not in Fife^AC/+ or RIM^ex103/+ compared with wild type (wild type, 32.44 ± 1.90 mV, n = 7 NMJs; Fife^AC/Fife^AC, 14.32 ± 2.29 mV, n = 11 NMJs, P < 0.0001; Fife^AC/Df, 11.37 ± 1.40 mV, n = 15 NMJs, P < 0.0001; RIM^ex103/RIM^ex73, 10.93 ± 1.35 mV, n = 8 NMJs, P < 0.0001; Fife^AC/+, 34.54 ± 2.12 mV, n = 9 NMJs, P = 0.99; RIM^ex103/+, 27.19 ± 3.06 mV, n = 12 NMJs, P = 0.74; Fife^AC+/+RIM^ex103, 12.94 ± 1.68 mV, n = 12 NMJs, P < 0.0001). Mean EJP amplitude is also significantly reduced in Fife^AC+/+RIM^ex103 compared with Fife^AC/+ (P < 0.0001) or RIM^ex103/+ (P < 0.0001). (H) Mean quantal content is also significantly reduced in Fife^AC/ Fife^AC, Fife^AC/Df, RIM^ex103/RIM^ex73, and Fife^AC+/+RIM^ex103, but not in Fife^AC/+ or RIM^ex103/+ compared with wild type (wild type, 32.56 ± 1.74, n = 7 NMJs; Fife^AC/Fife^AC, 16.59 ± 2.56, n = 11 NMJs, P < 0.0001; Fife^AC/Df, 13.22 ± 1.65, n = 15 NMJs, P < 0.0001; RIM^ex103/RIM^ex73, 11.80 ± 1.33, n = 8 NMJs, P < 0.0001; Fife^AC/+, 32.18 ± 1.81, n = 9 NMJs, P = 0.99; RIM^ex103/+, 24.04 ± 2.44, n = 12 NMJs, P = 0.75; Fife^AC+/+RIM^ex103, 11.55 ± 1.35 mV, n = 12 NMJs, P < 0.0001). Mean quantal content is also significantly reduced in Fife^AC+/+RIM^ex103 compared with Fife^AC/+ (P < 0.0001) or RIM^ex103/+ (P < 0.0001). ns, not significant; ***, P < 0.001, ****, P < 0.0001; analysis of variance followed by post hoc tests with Šidák correction. Error bars represent SEM.