Fig. 4. Phosphorylation of the MARCKS-homology domain partially inhibits Hts' ability to disrupt Dlg postsynaptic targeting to the larval NMJ.(A–D″) High.

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Fig. 4. Phosphorylation of the MARCKS-homology domain partially inhibits Hts' ability to disrupt Dlg postsynaptic targeting to the larval NMJ.(A–D″) High magnification views of a few boutons from third instar larval NMJs at muscles 6/7 in abdominal segment 4, immunostained with anti-Hrp (red) and anti-Dlg (green). Phosphorylation of the MARCKS-homology domain partially inhibits Hts' ability to disrupt Dlg postsynaptic targeting to the larval NMJ.(A–D″) High magnification views of a few boutons from third instar larval NMJs at muscles 6/7 in abdominal segment 4, immunostained with anti-Hrp (red) and anti-Dlg (green). (A–A″) Wild-type boutons show a tight association of Dlg to the postsynaptic membrane. (B–B″) Wild-type Hts expressed in the muscle with mef2-Gal4 disrupts Dlg postsynaptic targeting, as Dlg distribution appears diffuse and extends farther away from the NMJ. (C–C″) Non-phosphorylatable Hts disrupts Dlg postsynaptic targeting to a greater degree when compared to wild-type Hts, as Dlg distribution appears more diffuse and Dlg levels in the surrounding muscle are higher. (D–D″) In comparison to wild-type and non-phosphorylatable Hts, phospho-mimetic Hts disrupts Dlg postsynaptic targeting to a lesser degree, as Dlg distribution appears less diffuse. (E) Measurement of the ratio between Dlg and Hrp immunofluorescence surface area at the NMJ to quantitate the extent of Dlg spreading from the presynaptic membrane. The ratio for non-phosphorylatable Hts is significantly higher in comparison to wild-type Hts, indicating that blocking Hts phosphorylation enhances disruption of Dlg postsynaptic targeting. In contrast, the ratio for phospho-mimetic Hts is significantly lower when compared to wild-type Hts, indicating that Hts phosphorylation reduces disruption of Dlg postsynaptic targeting. The numbers on the bars of the graph indicate the number of NMJs evaluated for each genotype. As both NMJs in abdominal segment 4 of each body wall were examined, the number of larvae evaluated for each genotype is half. p values were determined using one-way ANOVA analyses followed by Tukey-Kramer post hoc tests. Asterisks in black show significance compared to the wild-type control. Asterisks in red show significance compared to transgenic wild-type Hts expression. *p < 0.05; **p < 0.01. Scale bar in Panel D″ represents 10 µm (for A–D″). Simon Ji Hau Wang et al. Biology Open 2014;bio.20148342 © 2014. Published by The Company of Biologists Ltd