Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology

Abstract

Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

Figure 1.

Decreased GluRIIA abundance at the third-instar NMJ in Pak mutant animals. A, Representative NMJ from wild-type (wt) and Pak mutant animals are shown that are costained with anti-GluRIIA and anti-HRP. At the Pak mutant NMJ, synaptic morphology is grossly normal, but GluRIIA abundance is substantially decreased. B, Quantification of the fluorescence intensity of anti-GluRIIA, anti-HRP, and anti-PAK staining at the NMJ of wild-type, control, and Pak mutant animals. Data are expressed as percentage of wild-type fluorescence intensity. The genotypes corresponding to each bar are shown below the graph. There is a statistically significant decrease in GluRIIA fluorescence intensity in the Pak mutant combinations compared with wild-type and control NMJ. Pak³/Df(3R)Win¹¹, 57 ± 2.3%, n = 17; Pak⁴/Df(3R)Win¹¹, 72 ± 4.4%, n = 17; Pak⁶/Df(3R)Win¹¹, 56 ± 3.8%, n = 18; and Pak³/Pak⁶, 72 ± 2.2%, n = 6. The average anti-HRP fluorescence varies <10% across all genotypes. Anti-Pak fluorescence is significantly decreased only in the Pak⁶/Df(3R)Win¹¹ combination compared with wild type (23.7 ± 4.7%). In all graphs, statistical significance is indicated as ^*p < 0.05; ^**p < 0.00005.

Figure 2.

Simultaneous disruption of Cdc42 and Rac in muscle leads to decreased GluRIIA levels. A, Wild-type (left) and transgenic larvae (right) that simultaneously overexpress dominant-negative Rac and dominant-negative Cdc42 [myosin heavy chain (MHC)-Gal4/UAS-Racⁿ¹⁷; UAS-Cdc42^N17/+] are shown stained with anti-HRP and anti-GluRIIA. B, GluRIIA levels are significantly decreased only in MHC-Gal4/UAS-Racⁿ¹⁷; UAS-Cdc42^N17/+, when both dominant-negative constructs are postsynaptically expressed (73.6 ± 3.3%; n = 17). Significance is denoted as ^*p < 0.0001.

Figure 3.

Decreased quantal size and normal homeostatic compensation at Pak mutant NMJ. A, Quantification of quantal size in control (filled bar) and experimental genotypes (open bars). Wild-type and heterozygous controls [Df(3R)Win¹¹/+ and Pak⁶/+; GluRIIA^SP16/+] have quantal sizes equal to 1.1 ± 0.1 mV (n = 6), 0.98 ± 0.7 mV (n = 6), and 0.96 ± 0.83 mV (n = 5), respectively. Experimental genotypes all showed significant decreases in quantal size: Pak⁶/Df(3R)Win¹¹, 0.73 ± 0.07 mV, n = 6; GluRIIA^SP16, 0.51 ± 0.01 mV, n = 18; GluRIIA^SP16; Pak⁶/+, 0.46 ± 0.02 mV, n = 5; GluRIIA^SP16; Pak³/Pak⁶, 0.4 ± 0.01, n = 7. There is also a small, yet statistically significant, difference between GluRIIA^SP16 and GluRIIA^SP16; Pak³/Pak⁶. B, Quantification of quantal content in control (filled bars) and experimental genotypes (open bars). There is no difference in quantal content comparing the experimental genotype Pak⁶/Df(3R) Win¹¹ with wildtype or experimental controls. Values are as follows: wildtype, 34.5 ± 1.5, n = 6; Df(3R)Win¹¹/+, 32.1 ± 2.8, n = 6; Pak⁶/+; GluRIIA^SP16/+, 39.3 ± 5.2, n = 5; Pak⁶/Df(3R)Win¹¹, 40.7 ± 3.7, n = 6. The experimental genotypes GluRIIA^SP16, GluRIIA^SP16; Pak⁶/+, and GluRIIA^SP16; Pak³/Pak⁶ all showed significant increases in quantal content compared with wild type and genetic controls, indicating that homeostatic compensation has occurred. Values are as follows: GluRIIA^SP16, 58 ± 3.7 mV, n = 15; GluRIIA^SP16; Pak⁶/+, 48.8 ± 5.2 mV, n = 5; and GluRIIA^SP16; Pak³/Pak⁶, 63.6 ± 8.7, n = 6. C, Representative traces of evoked potentials (left; each trace represents the average of 10 individual traces) and spontaneous miniature potentials (right) from control [Df(3R)Win¹¹/+] and Pak mutant NMJ [Pak⁶/Df(3R)Win¹¹]. The traces show the reduction in quantal size in Pak mutant animals and the wild-type EPSP amplitude indicative of effective synaptic homeostasis. Calibration: 10 mV, 50 msec (for evoked release); 1 mV, 250 msec (for spontaneous traces). Significance is denoted as ^*p < 0.05; ^**p < 0.002; ^***p < 0.00002.

Figure 4.

Synaptic localization of Dock. A wild-type (left) and a dock mutant NMJ [right; dock^P1/Df(2L)ast²] are shown stained with anti-HRP and anti-Dock. Anti-Dock immunoreactivity is localized to the NMJ in wild type. In dock null animals, anti-Dock staining is completely absent from the NMJ (top right). The images shown are calibrated identically.

Figure 5.

Synaptic localization of GluRIIA is decreased in dock mutants. A, Representative images of a wild-type (left) and a dock null NMJ [dock^P1/Df(2L)ast²] stained with anti-GluRIIA, anti-Pak, and anti-HRP. GluRIIA staining is reduced in the dock mutant animals without a corresponding decrease in Pak levels at the synapse. B, Quantification of the fluorescence intensity of anti-GluRIIA, anti-HRP, and anti-PAK staining. There is a significant decrease in the intensity of GluRIIA staining in dock^P1/Df(2L)ast² null animals (61.2 ± 3.6%; n = 8) and dock4/Df(2L)ast² (64.3 ± 4.8%; n = 6) compared with wild-type and the heterozygous controls dock^P1/+, dock⁴/+, and Df(2L)ast²/+. Additionally, we observed a significant increase in GluRIIA staining and Pak staining in the dock^P1/+ animals (^*p < 0.05; ^**p < 0.0002).

Figure 6.

Dock and Pak are synaptically localized independently of each other. A, Representative images of a wild-type (left) and a dock⁴/Df(2L)ast² NMJ stained with anti-dock and anti-HRP. Dock protein, with a point mutation in the SH2 domain, is no longer highly localized at the synapse. B, Representative images of wild-type (left) and a dock^P1/Df(2L)ast² NMJ stained with anti-Pak. Pak is localized properly in dock null mutants. C, Representative images of wild-type (left) and Pak⁴/Df(3R)Win¹¹ NMJ stained with anti-dock. Dock is localized properly in Pak mutants.

Figure 7.

Dock and Pak interact genetically to regulate synaptic GluRIIA levels. Quantification of anti-GluRIIA and anti-Pak fluorescent intensities in Pak and dock double-mutant combinations is shown. Compared with wild type, the dock^P1/+; Pak⁶/+ transheterozygous animals show a decrease in GluRIIA staining at the synapse (72 ± 2.2%; n = 6). A further reduction in GluRIIA staining is seen in a dock null animal with one copy of a Pak mutant gene, dock^P1/Df(2L)ast²; Pak⁶/+ (49 ± 3.0%; n = 6), and in the Pak mutant animal with one mutant copy of dock, dock^P1/+; Pak⁶/Df(3L)Win¹¹ (57 ± 2.4%; n = 6). For comparison, bars representing the single mutants, dock^P1/Df(2L)ast² and Pak⁶/Df(3L)Win¹¹, are included from Figures 2 and 5. As an additional control, Pak⁶/+ animals are shown, which have a reduction in GluRIIA levels (89 ± 2.2%; n = 7). Levels of significance are ^*p < 0.05; ^**p < 0.0005; ^***p < 0.000005.

Figure 8.

Synaptic localization of Dlg is decreased in dock and Pak mutants. A, Representative images of wild-type (left), Pak⁴/Df(3R)Win¹¹ (center), and Pak³/Df(3R)Win¹¹ (right) NMJs stained with anti-Dlg. Dlg staining is severely reduced only in the Pak kinase domain point mutation (Pak³) and not the dock interaction domain point mutation (Pak⁴). B, Quantification of the fluorescence intensity of anti-Dlg staining in Pak and dock mutants. There is a strong decrease in the intensity of Dlg staining in Pak³/Df(3R)Win¹¹ (50.5 ± 2.0%; n = 16) and Pak⁵/Df(3R)Win¹¹ (55.5 ± 2.9%; n = 6) animals compared with wild type. There is a smaller decrease in intensity of Dlg staining in Pak⁴/Df(3R)Win¹¹ animals (79.1 ± 2.4%; n = 11), which is significantly different from the decrease in Pak⁵/Df(3R)Win¹¹ animals as indicated in the graph. Dlg intensity among dock heterozygous controls exhibits a small yet significant decrease compared with wild type (dock⁴/+, 89.43 ± 2.5%, n = 9; Df(2L)ast²/+, 86 ± 2.7%, n = 8). dock⁴/Df(2L)ast² NMJs also exhibit a small yet significant decrease in Dlg staining compared with wild-type (80.8 ± 3.0%; n = 10) NMJs. Compared with heterozygous controls, as indicated on the graph, there is no significant decrease in Dlg staining in dock mutant synapses. Levels of significance are ^*p < 0.005; ^**p < 0.0000001.

Figure 9.

The postsynaptic Pix-Pak-Dock signaling system. Pix localizes Pak to the synaptic membrane. After activation, Pak signaling diverges. In one branch, Pak binds to Dock. Dock itself is recruited to the synapse via an essential SH2-mediated interaction with an unknown synaptic protein. Dock-Pak binding is required for normal GluR abundance. Pak signaling also diverges to control Dlg levels and thereby regulates the formation of the postsynaptic muscle membrane folds.