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Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

Wang SJ, Tsai A, Wang M, Yoo S, Kim HY, Yoo B, Chui V, Kisiel M, Stewart B, Parkhouse W, Harden N, Krieger C - Biol Open (2014)

Bottom Line: We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle.We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain.These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.

No MeSH data available.


Phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.(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 1B1 (green). (A–A″) Wild-type boutons showing endogenous, postsynaptic Hts distribution. (B–B″) Wild-type Hts, HtsS704S, expressed in the muscle with mef2-Gal4 concentrates at the postsynaptic membrane around the boutons. (C–C″) Non-phosphorylatable Hts, HtsS704A, also concentrates at the postsynaptic membrane. (D–D″) In contrast to wild-type and non-phosphorylatable Hts, phospho-mimetic Hts, HtsS704D, does not specifically concentrate at the postsynaptic membrane. (E) Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity to quantitate Hts localization at the NMJ. The ratio for phospho-mimetic Hts is significantly lower in comparison to wild-type Hts, indicating that phospho-mimetic Hts does not localize as well to the NMJ. 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. Hrp was used as an internal control. p values were determined using one-way ANOVA analyses followed by Tukey-Kramer post hoc tests. *p < 0.01. Scale bar in Panel D″ represents 10 µm (for A–D″).
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f02: Phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.(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 1B1 (green). (A–A″) Wild-type boutons showing endogenous, postsynaptic Hts distribution. (B–B″) Wild-type Hts, HtsS704S, expressed in the muscle with mef2-Gal4 concentrates at the postsynaptic membrane around the boutons. (C–C″) Non-phosphorylatable Hts, HtsS704A, also concentrates at the postsynaptic membrane. (D–D″) In contrast to wild-type and non-phosphorylatable Hts, phospho-mimetic Hts, HtsS704D, does not specifically concentrate at the postsynaptic membrane. (E) Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity to quantitate Hts localization at the NMJ. The ratio for phospho-mimetic Hts is significantly lower in comparison to wild-type Hts, indicating that phospho-mimetic Hts does not localize as well to the NMJ. 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. Hrp was used as an internal control. p values were determined using one-way ANOVA analyses followed by Tukey-Kramer post hoc tests. *p < 0.01. Scale bar in Panel D″ represents 10 µm (for A–D″).

Mentions: PKC-dependent phosphorylation causes adducin to translocate from the actin-spectrin cytoskeleton at cell-cell adhesion sites to the cytoplasm in epithelial cells (Matsuoka et al., 1998). Moreover, adducin is phosphorylated by PKC at elevated levels in renal carcinomas, and this aberrant phosphorylation correlates with changes in adducin subcellular distribution (Fowler et al., 1998). We assessed whether phosphorylation of the MARCKS-homology domain also regulates Hts localization at the postsynaptic membrane of larval NMJs. This was done by examining the distribution of wild-type, non-phosphorylatable and phospho-mimetic Hts when expressed in the muscle with mef2-Gal4. Both wild-type and non-phosphorylatable Hts concentrated at the postsynaptic membrane of NMJs (Fig. 2B–C″). Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity, which allowed us to quantitate Hts localization at the NMJ, revealed no significant difference between the two versions of Hts (Fig. 2E). However, phospho-mimetic Hts levels at the postsynaptic membrane were significantly reduced in comparison to wild-type and non-phosphorylatable Hts (Fig. 2D–E). The ratio was around 1, indicating that phospho-mimetic Hts does not specifically concentrate at the NMJ. These results show that phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.


Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

Wang SJ, Tsai A, Wang M, Yoo S, Kim HY, Yoo B, Chui V, Kisiel M, Stewart B, Parkhouse W, Harden N, Krieger C - Biol Open (2014)

Phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.(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 1B1 (green). (A–A″) Wild-type boutons showing endogenous, postsynaptic Hts distribution. (B–B″) Wild-type Hts, HtsS704S, expressed in the muscle with mef2-Gal4 concentrates at the postsynaptic membrane around the boutons. (C–C″) Non-phosphorylatable Hts, HtsS704A, also concentrates at the postsynaptic membrane. (D–D″) In contrast to wild-type and non-phosphorylatable Hts, phospho-mimetic Hts, HtsS704D, does not specifically concentrate at the postsynaptic membrane. (E) Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity to quantitate Hts localization at the NMJ. The ratio for phospho-mimetic Hts is significantly lower in comparison to wild-type Hts, indicating that phospho-mimetic Hts does not localize as well to the NMJ. 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. Hrp was used as an internal control. p values were determined using one-way ANOVA analyses followed by Tukey-Kramer post hoc tests. *p < 0.01. Scale bar in Panel D″ represents 10 µm (for A–D″).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265757&req=5

f02: Phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.(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 1B1 (green). (A–A″) Wild-type boutons showing endogenous, postsynaptic Hts distribution. (B–B″) Wild-type Hts, HtsS704S, expressed in the muscle with mef2-Gal4 concentrates at the postsynaptic membrane around the boutons. (C–C″) Non-phosphorylatable Hts, HtsS704A, also concentrates at the postsynaptic membrane. (D–D″) In contrast to wild-type and non-phosphorylatable Hts, phospho-mimetic Hts, HtsS704D, does not specifically concentrate at the postsynaptic membrane. (E) Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity to quantitate Hts localization at the NMJ. The ratio for phospho-mimetic Hts is significantly lower in comparison to wild-type Hts, indicating that phospho-mimetic Hts does not localize as well to the NMJ. 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. Hrp was used as an internal control. p values were determined using one-way ANOVA analyses followed by Tukey-Kramer post hoc tests. *p < 0.01. Scale bar in Panel D″ represents 10 µm (for A–D″).
Mentions: PKC-dependent phosphorylation causes adducin to translocate from the actin-spectrin cytoskeleton at cell-cell adhesion sites to the cytoplasm in epithelial cells (Matsuoka et al., 1998). Moreover, adducin is phosphorylated by PKC at elevated levels in renal carcinomas, and this aberrant phosphorylation correlates with changes in adducin subcellular distribution (Fowler et al., 1998). We assessed whether phosphorylation of the MARCKS-homology domain also regulates Hts localization at the postsynaptic membrane of larval NMJs. This was done by examining the distribution of wild-type, non-phosphorylatable and phospho-mimetic Hts when expressed in the muscle with mef2-Gal4. Both wild-type and non-phosphorylatable Hts concentrated at the postsynaptic membrane of NMJs (Fig. 2B–C″). Measurement of the ratio between synaptic and extrasynaptic Hts immunofluorescence intensity, which allowed us to quantitate Hts localization at the NMJ, revealed no significant difference between the two versions of Hts (Fig. 2E). However, phospho-mimetic Hts levels at the postsynaptic membrane were significantly reduced in comparison to wild-type and non-phosphorylatable Hts (Fig. 2D–E). The ratio was around 1, indicating that phospho-mimetic Hts does not specifically concentrate at the NMJ. These results show that phosphorylation of the MARCKS-homology domain disrupts Hts localization at the postsynaptic membrane of larval NMJs.

Bottom Line: We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle.We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain.These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.

No MeSH data available.