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. 2016 Feb 7;25(8):1528–1542. doi: 10.1093/hmg/ddw031

Figure 6.

Figure 6.

Functional analysis of C236R, G319R and G605R mutations using garss266/s266 larvae. (A) Native western blot analysis of protein extracts from injected with wild-type, C236R and G605R zGars and uninjected mutant embryos at 72 hpf showed that in injected garss266/s266 embryos, Gars existed in an active, dimer formation, while in the control as a monomer. In contrast, injection with G319R did not result in the formation of heterodimers. (B–D). Projections of confocal z-stacks at the level of hindgut extension of 96 hpf. (E–G) During the development of garss266/s266, denervated post-synaptic regions were observed, as only the 5.5% ± 0.8% of synaptic occupied areas were present in mutant larvae. (H–J) Forty-eight percent of the homozygous mutant larvae injected with wild-type zGars exhibited blood flow (n = 41, not shown) at 6 dpf and showed nearly wild-type levels of synaptic areas at 96 hpf. (K–M) Seventeen percent of the homozygous mutant larvae injected with C236R mRNA exhibit blood flow (n = 128, not shown) at 6 dpf and showed an increase of synaptic areas up to 2-fold at 96 hpf compared with mutant control. (N–P) Homozygous mutant larvae injected with G319R mRNA (n = 35). Injected embryos had denervated muscles with a similar decrease of NMJs occupied areas compared with garss266/s266 uninjected larvae. (Q–S) Homozygous mutant larvae (n = 47) injected with G605R mRNA. Injected embryos had denervated muscles with a similar decrease of NMJs density compared with garss266/s266 uninjected larvae. Although dimers formed in the injected larvae, both mutations rendered the dimer non-functional and as a result embryos died by 5 dpf. (S and T) Quantification of synaptic occupied areas in injected larvae (wild-type n = 10, garss266/s266 n = 14, garss266/s266 wild-type zGars n = 10, garss266/s266 C157R zGars n = 9, garss266/s266 G240R zGars n = 10, garss266/s266 G526R zGars n = 11, ***P < 0.0001, one-way ANOVA). (U) Quantification of co-localization of SV2 and aBTX in injected embryos (wild-typen = 10, garss266/s266 n = 14, garss266/s266 wild-type zGars n = 10, garss266/s266 C236R zGars n = 9, garss266/s266 G319R zGars n = 10, garss266/s266 G605R zGars n = 11, ***P < 0.0001, one-way ANOVA). Scale bars: 25 μm.