S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development

Abstract

The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.

Figure 1.

Presynaptic S6K regulates bouton size without altering bouton number. (A–F) Third instar NMJs on muscle 4 of segment A3 were visualized with the synaptic vesicle marker antisynapsin (green) and neuronal membrane marker HRP (magenta). Twofold magnifications from selected regions are shown in insets. Wild type (WT; A), S6K^l-1/Df(3L)CH18 (B), C155/+;UAS-S6K/+;S6K^l-1/Df(3L)CH18 (C), UAS-S6K/+;S6K^l-1,BG57/Df(3L)CH18 (D), C155/+;UAS-S6K^STDETE/+;S6K^l-1/Df(3L)CH18 (E), and C155/+;UAS-S6K^KQ/+;S6K^l-1/Df(3L)CH18 (F) are shown. (G–I) Quantification of bouton size (G and I) and bouton number (H). n > 10 for all genotypes. *, P < 0.05; ***, P < 0.0001. Error bars show SEM. Bar, 10 µm.

Figure 2.

S6K localizes to presynaptic active zones. (A–D) Z-series projection of OK371-Gal4/+;UAS-3×FLAG-S6K/+ NMJs at muscle 4. (A and B) Synapses are colabeled with anti-FLAG (S6K) and anti-Brp. (B) Higher magnification images from NMJ shown in A. (C) Synapses are colabeled with anti-FLAG (S6K) and anti-GluRIIC. (D) Synapses are colabeled with anti-FLAG (S6K) and anti-Dap160. (E) Z-series projection of OK371-Gal4,Brp⁶⁹/Brp⁶⁹;UAS-Venus-S6K/+ synapses. Venus (S6K) and anti-Brp (absent in brp mutant) are shown. (F) Z-series projection of C155/+;Rab3^rup/Rab3^rup;UAS-Venus-S6K/+ synapses. Venus (S6K) and anti-Brp are shown. Bars: (A) 10 µm; (B–F) 2 µm.

Figure 3.

S6K regulates active zone number and density. (A–D) NMJs on muscle 4 of segment A3 are labeled with the active zone marker anti-Brp and neuronal membrane marker HRP. Insets represent higher magnification images of select synaptic boutons within each image. Wild type (WT; A), S6K^l-1/Df(3L)CH18 (B), C155/+;UAS-S6K/+;S6K^l-1/Df(3L)CH18 (C), and C155/+;UAS-S6K^STDETE/+;S6K^l-1/Df(3L)CH18 (D) are shown. (E–G) Quantification of active zone number (E), synaptic area (F), and active zone density (G). n > 10 for all genotypes. *, P < 0.05; ***, P < 0.0001. Error bars show SEM. Bars, 10 µm.

Figure 4.

Altered synaptic function at S6K mutant NMJ. (A) Recordings are made from the NMJ at muscle 6, and data are shown for the mean mEPSP amplitude, mean EPSP amplitude, and mean quantal content (EPSP/mEPSP) and muscle input resistance (Rin). Genotypes in A include wild type (WT), S6K, and S6K/Df (S6K^l-1/Df(3L)^CH18). (B) Data are normalized to each genotype in the absence of PhTx as performed previously (Frank et al., 2006, 2009). Application of PhTx to the NMJ reduces mEPSP amplitudes and causes a significant increase in quantal content (Qc) compared with baseline (100%). (C) Extracellular calcium concentration is plotted against quantal content for the indicated genotypes. (D) Acute expression of UAS-S6K^STDETE with elav-Geneswitch during the last 48 h of larval development is without effect on quantal content. Error bars show SEM.

Figure 5.

PDK1 localizes to the Drosophila NMJs. (A–D) Z-series projection of OK371-Gal4/+;UAS-3×FLAG-PDK1/+ NMJs on muscle 4. (A) Synapses are colabeled with anti-FLAG (PDK1) and anti-Brp. (B) Higher magnification images of what is shown in A. (C) Synapses are colabeled with anti-FLAG (PDK1) and anti-GluRIIC. (D) Synapses are colabeled with anti-FLAG (PDK1) and anti-Dap160. Bars: (A) 10 µm; (B–D) 2 µm.

Figure 6.

PDK1 regulates bouton size, bouton number, and muscle size. (A–E) Third-instar NMJs on muscle 4 of segment A3 are visualized with the synaptic vesicle marker antisynapsin (green) and neuronal membrane marker HRP (magenta). Twofold magnifications from selected regions are shown in insets. Wild type (WT; A), PDK1³³/PDK1³³ (B), OK371-Gal4/UAS-PDK1;PDK1³³/PDK1³³ (C), UAS-PDK1/+;BG57-Gal4,PDK1³³/PDK1³³ (D), and UAS-PDK1/+;Tubulin-Gal4,PDK1³³/PDK1³³ (E) are shown. (F–I) Quantification of bouton size (F), muscle size (G), absolute bouton number (H), and normalized bouton number (I). OK371 overexpression (O/E): OK371-Gal4/UAS-PDK1. BG57 overexpression: UAS-PDK1/+;BG57-Gal4/+. n > 10 for all genotypes. ***, P < 0.0001. Error bars show SEM. Bar, 10 µm.

Figure 7.

PDK1 regulates active zone number and density. (A–D) NMJs on muscle 4 of segment A3 are labeled with the active zone marker anti-Brp and neuronal membrane marker HRP. Insets represent higher magnification images of select synaptic boutons within each image. Wild type (WT; A), PDK1³³/PDK1³³ (B), OK371-Gal4/UAS-PDK1;PDK1³³/PDK1³³ (C), and OK371-Gal4/UAS-PDK1 (D) are shown. (E–G) Quantification of active zone number (E), synaptic area (F), and active zone density (G). n > 10 for all genotypes. O/E, overexpression. **, P < 0.01; ***, P < 0.0001. Error bars show SEM. Bar, 10 µm.

Figure 8.

Neuronal overexpression of active S6K is able to rescue bouton size defects in PDK1 mutants. (A–C) Third instar NMJs on muscle 4 of segment A3 are visualized with the synaptic vesicle marker antisynapsin and neuronal membrane marker HRP. Twofold magnifications from selected regions are shown in insets. OK371-Gal4/UAS-S6K;PDK1³³/PDK1³³ (A), OK371-Gal4/UAS-S6K^STDETE;PDK1³³/PDK1³³ (B), and OK371-Gal4/UAS-PDK1;S6K^l-1/Df(3L)CH18 (C) are shown. (D and E) Quantification of bouton size (D) and bouton number (E). n > 10 for all genotypes. **, P < 0.01; ***, P < 0.0001. Error bars show SEM. Bar, 10 µm.

Figure 9.

Simultaneous overexpression of S6K and PDK1 increases bouton size and bouton number. (A–F) Third instar NMJs on muscle 4 of segment A3 are visualized with the synaptic vesicle marker antisynapsin and neuronal membrane marker HRP. Twofold magnifications from selected regions are shown in insets. Wild type (WT; A), OK371-Gal4/+;UAS-PDK1/+ (B), OK371-Gal4/UAS-S6K (C), OK371-Gal4/UAS-S6K^STDETE (D), OK371-Gal4/UAS-S6K;UAS-PDK1/+ (E), and OK371-Gal4/UAS- S6K^STDETE;UAS-PDK1/+ (F) are shown. (G and H) Quantification of bouton size (G) and bouton number (H). n > 10 for all genotypes. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Error bars show SEM. Bar, 10 µm.