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The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development.

Mosca TJ, Schwarz TL - Nat. Neurosci. (2010)

Bottom Line: The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain.We found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large.Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway.

View Article: PubMed Central - PubMed

Affiliation: The F.M. Kirby Neurobiology Center, Children's Hospital Boston, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Synapse-to-nucleus signaling is critical for synaptic development and plasticity. In Drosophila, the ligand Wingless causes the C terminus of its Frizzled2 receptor (Fz2-C) to be cleaved and translocated from the postsynaptic density to nuclei. The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain. We found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large. These developmental defects in imp-beta11 mutants could be overcome by expression of Fz2-C fused to a nuclear localization sequence that can bypass Importin-beta11. Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway.

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Ghost Boutons are More Frequent at importin-β11 and -α2 Mutant NMJs(a–i) Antibodies to HRP (magenta) and Discs Large (DLG, green) marked pre- and postsynaptic compartments of NMJs at muscles 6 and 7 of the indicated genotypes. Typically, each wild-type presynaptic bouton correctly faces a postsynaptic concentration of DLG but, particularly in the mutant genotypes, there is an increased frequency of presynaptic boutons that have no opposite postsynaptic DLG (arrows). At other mutant boutons, DLG staining is weaker but still present. Wild-type = (y,w; FRT42D; +; +), importin-β11 = (y,w; imp-β1170 / Df; +; +), importin-α2 mutant = (y,w; imp-α2D14; +; +) Scale bar = 5 µm. (j) Loss of either Importin-β11 or Importin-α2 caused a threefold increase in ghost boutons. The frequency of ghost boutons could be returned to control levels by expression of the particular importin in the muscle, but not neurons. Also, muscle expression of NLS-Fz2-C rescued the importin-β11 phenotype but a control NLS-tagged GFP transgene did not. Ghost boutons were quantified; genotypes (as indicated by colors) and specific values are as follows: +/+ (y,w; FRT42D; +; +) = 1.0 ± 0.18 ghost boutons / NMJ, n = 10 animals, 59 NMJs; β11 −/− (y,w; imp-β1170 / Df; +; +) = 3.1 ± 0.4 n = 13 animals, 78 NMJs; α2 −/− (y,w; imp-α2D14; +; +) = 3.1 ± 0.4, n = 11 animals, 65 NMJs, p vs. imp-β1170 / Df >0.8; wit −/− (y,w; +; witA12/B11; +) = 0.71 ± 0.14, n = 11 animals, 65 NMJs, p vs. +/+ > 0.2; β11 −/− Neuron β11 (y,w; imp-β1170 / Df; elav-GAL4 / UAS-importin-β11-eGFP; +) = 3.2 ± 0.39 ghost boutons per NMJ, n = 8 animals, 48 NMJs, p vs. β11 −/− > 0.8; β11 −/− Muscle β11 (y,w; imp-β1170 / Df; 24B–GAL4 / UAS- importin-β11-eGFP; +) = 1.1 ± 0.17, n = 11 animals, 65 NMJs, p vs. +/+ > 0.5, vs. imp-β1170 / Df < 0.0001; α2 −/− Neuron α2 (y,w; imp-α2D14; elav-GAL4 / UAS-importin-α2; +) = 3.2 ± 0.33, n = 10 animals, 57 NMJs, p vs. α2−/− > 0.4; α2 −/− Muscle α2 (y,w; imp-α2D14; 24B–GAL4 / UAS-importin-α2; +) = 1.0 ± 0.19, n = 9 animals, 52 NMJs, p vs. +/+ > 0.8, vs. imp-α2D14 < 0.0001; β11 −/− Muscle Ketel = y,w; imp-β1170 / Df; 24B–GAL4 / UAS-Ketel; + (3.1 ± 0.38, n = 7 animals, 40 NMJs, p vs. β11 −/− >0.7; β11 −/− Muscle NLS-Fz2-C (w, UAS-myc-NLS-DFz2-C / + or Y; imp-β1170 / Df; 24B–GAL4 / +; +) = 1.1 ± 0.16 ghost boutons per NMJ, n = 14 animals, 78 NMJs, p vs. +/+ > 0.7, vs. imp-β1170 / Df < 0.0001; β11 −/− Muscle NLS-GFP (y,w; imp-β1170 / Df; UAS-NLS-GFP / 24B–GAL4; +) = 3.0 ± 0.48, n = 8 animals, 45 NMJs, p vs. imp-β1170 / Df > 0.4. *** p < 0.0001 vs. + / +.
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Figure 6: Ghost Boutons are More Frequent at importin-β11 and -α2 Mutant NMJs(a–i) Antibodies to HRP (magenta) and Discs Large (DLG, green) marked pre- and postsynaptic compartments of NMJs at muscles 6 and 7 of the indicated genotypes. Typically, each wild-type presynaptic bouton correctly faces a postsynaptic concentration of DLG but, particularly in the mutant genotypes, there is an increased frequency of presynaptic boutons that have no opposite postsynaptic DLG (arrows). At other mutant boutons, DLG staining is weaker but still present. Wild-type = (y,w; FRT42D; +; +), importin-β11 = (y,w; imp-β1170 / Df; +; +), importin-α2 mutant = (y,w; imp-α2D14; +; +) Scale bar = 5 µm. (j) Loss of either Importin-β11 or Importin-α2 caused a threefold increase in ghost boutons. The frequency of ghost boutons could be returned to control levels by expression of the particular importin in the muscle, but not neurons. Also, muscle expression of NLS-Fz2-C rescued the importin-β11 phenotype but a control NLS-tagged GFP transgene did not. Ghost boutons were quantified; genotypes (as indicated by colors) and specific values are as follows: +/+ (y,w; FRT42D; +; +) = 1.0 ± 0.18 ghost boutons / NMJ, n = 10 animals, 59 NMJs; β11 −/− (y,w; imp-β1170 / Df; +; +) = 3.1 ± 0.4 n = 13 animals, 78 NMJs; α2 −/− (y,w; imp-α2D14; +; +) = 3.1 ± 0.4, n = 11 animals, 65 NMJs, p vs. imp-β1170 / Df >0.8; wit −/− (y,w; +; witA12/B11; +) = 0.71 ± 0.14, n = 11 animals, 65 NMJs, p vs. +/+ > 0.2; β11 −/− Neuron β11 (y,w; imp-β1170 / Df; elav-GAL4 / UAS-importin-β11-eGFP; +) = 3.2 ± 0.39 ghost boutons per NMJ, n = 8 animals, 48 NMJs, p vs. β11 −/− > 0.8; β11 −/− Muscle β11 (y,w; imp-β1170 / Df; 24B–GAL4 / UAS- importin-β11-eGFP; +) = 1.1 ± 0.17, n = 11 animals, 65 NMJs, p vs. +/+ > 0.5, vs. imp-β1170 / Df < 0.0001; α2 −/− Neuron α2 (y,w; imp-α2D14; elav-GAL4 / UAS-importin-α2; +) = 3.2 ± 0.33, n = 10 animals, 57 NMJs, p vs. α2−/− > 0.4; α2 −/− Muscle α2 (y,w; imp-α2D14; 24B–GAL4 / UAS-importin-α2; +) = 1.0 ± 0.19, n = 9 animals, 52 NMJs, p vs. +/+ > 0.8, vs. imp-α2D14 < 0.0001; β11 −/− Muscle Ketel = y,w; imp-β1170 / Df; 24B–GAL4 / UAS-Ketel; + (3.1 ± 0.38, n = 7 animals, 40 NMJs, p vs. β11 −/− >0.7; β11 −/− Muscle NLS-Fz2-C (w, UAS-myc-NLS-DFz2-C / + or Y; imp-β1170 / Df; 24B–GAL4 / +; +) = 1.1 ± 0.16 ghost boutons per NMJ, n = 14 animals, 78 NMJs, p vs. +/+ > 0.7, vs. imp-β1170 / Df < 0.0001; β11 −/− Muscle NLS-GFP (y,w; imp-β1170 / Df; UAS-NLS-GFP / 24B–GAL4; +) = 3.0 ± 0.48, n = 8 animals, 45 NMJs, p vs. imp-β1170 / Df > 0.4. *** p < 0.0001 vs. + / +.

Mentions: To test directly for an effect of the wg pathway and specifically Fz2-C nuclear import, we immunostained wg and dfz2 larvae with anti-HRP and anti-DLG (Supplementary Fig. 5) and counted instances at muscles 6 and 7 where boutons lacked surrounding DLG. In wild-type, ghost boutons were occasionally seen (Figure 6j) and found to lack apposite glutamate receptors (by GluRIIC staining) and the active zone protein Bruchpilot, but contained Synaptotagmin I immunoreactivity. When Wg / Fz2 signalling was perturbed, ghost bouton frequency increased: in hypomorphic wg1 mutants, their frequency doubled while in the more severe wgTS mutant, when shifted to a non-permissive temperature, their frequency was increased 4-fold (Supplementary Fig. 5). Similarly, in dfz2 mutants, ghost boutons increased nearly 3-fold (Supplementary Fig. 5). Neuronal overexpression of Wingless (Fig. 2)10 and hyperactivity mutations like eag1ShKS133 double mutants12 increase Wg signaling and nuclear Fz2-C. In these genotypes, ghost boutons were significantly fewer than in wild-type (Supplementary Fig. 5). Thus manipulations that decreased Wg / Fz2 signaling increased the frequency of ghost boutons and those that enhanced signaling diminished their occurrence.


The nuclear import of Frizzled2-C by Importins-beta11 and alpha2 promotes postsynaptic development.

Mosca TJ, Schwarz TL - Nat. Neurosci. (2010)

Ghost Boutons are More Frequent at importin-β11 and -α2 Mutant NMJs(a–i) Antibodies to HRP (magenta) and Discs Large (DLG, green) marked pre- and postsynaptic compartments of NMJs at muscles 6 and 7 of the indicated genotypes. Typically, each wild-type presynaptic bouton correctly faces a postsynaptic concentration of DLG but, particularly in the mutant genotypes, there is an increased frequency of presynaptic boutons that have no opposite postsynaptic DLG (arrows). At other mutant boutons, DLG staining is weaker but still present. Wild-type = (y,w; FRT42D; +; +), importin-β11 = (y,w; imp-β1170 / Df; +; +), importin-α2 mutant = (y,w; imp-α2D14; +; +) Scale bar = 5 µm. (j) Loss of either Importin-β11 or Importin-α2 caused a threefold increase in ghost boutons. The frequency of ghost boutons could be returned to control levels by expression of the particular importin in the muscle, but not neurons. Also, muscle expression of NLS-Fz2-C rescued the importin-β11 phenotype but a control NLS-tagged GFP transgene did not. Ghost boutons were quantified; genotypes (as indicated by colors) and specific values are as follows: +/+ (y,w; FRT42D; +; +) = 1.0 ± 0.18 ghost boutons / NMJ, n = 10 animals, 59 NMJs; β11 −/− (y,w; imp-β1170 / Df; +; +) = 3.1 ± 0.4 n = 13 animals, 78 NMJs; α2 −/− (y,w; imp-α2D14; +; +) = 3.1 ± 0.4, n = 11 animals, 65 NMJs, p vs. imp-β1170 / Df >0.8; wit −/− (y,w; +; witA12/B11; +) = 0.71 ± 0.14, n = 11 animals, 65 NMJs, p vs. +/+ > 0.2; β11 −/− Neuron β11 (y,w; imp-β1170 / Df; elav-GAL4 / UAS-importin-β11-eGFP; +) = 3.2 ± 0.39 ghost boutons per NMJ, n = 8 animals, 48 NMJs, p vs. β11 −/− > 0.8; β11 −/− Muscle β11 (y,w; imp-β1170 / Df; 24B–GAL4 / UAS- importin-β11-eGFP; +) = 1.1 ± 0.17, n = 11 animals, 65 NMJs, p vs. +/+ > 0.5, vs. imp-β1170 / Df < 0.0001; α2 −/− Neuron α2 (y,w; imp-α2D14; elav-GAL4 / UAS-importin-α2; +) = 3.2 ± 0.33, n = 10 animals, 57 NMJs, p vs. α2−/− > 0.4; α2 −/− Muscle α2 (y,w; imp-α2D14; 24B–GAL4 / UAS-importin-α2; +) = 1.0 ± 0.19, n = 9 animals, 52 NMJs, p vs. +/+ > 0.8, vs. imp-α2D14 < 0.0001; β11 −/− Muscle Ketel = y,w; imp-β1170 / Df; 24B–GAL4 / UAS-Ketel; + (3.1 ± 0.38, n = 7 animals, 40 NMJs, p vs. β11 −/− >0.7; β11 −/− Muscle NLS-Fz2-C (w, UAS-myc-NLS-DFz2-C / + or Y; imp-β1170 / Df; 24B–GAL4 / +; +) = 1.1 ± 0.16 ghost boutons per NMJ, n = 14 animals, 78 NMJs, p vs. +/+ > 0.7, vs. imp-β1170 / Df < 0.0001; β11 −/− Muscle NLS-GFP (y,w; imp-β1170 / Df; UAS-NLS-GFP / 24B–GAL4; +) = 3.0 ± 0.48, n = 8 animals, 45 NMJs, p vs. imp-β1170 / Df > 0.4. *** p < 0.0001 vs. + / +.
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Figure 6: Ghost Boutons are More Frequent at importin-β11 and -α2 Mutant NMJs(a–i) Antibodies to HRP (magenta) and Discs Large (DLG, green) marked pre- and postsynaptic compartments of NMJs at muscles 6 and 7 of the indicated genotypes. Typically, each wild-type presynaptic bouton correctly faces a postsynaptic concentration of DLG but, particularly in the mutant genotypes, there is an increased frequency of presynaptic boutons that have no opposite postsynaptic DLG (arrows). At other mutant boutons, DLG staining is weaker but still present. Wild-type = (y,w; FRT42D; +; +), importin-β11 = (y,w; imp-β1170 / Df; +; +), importin-α2 mutant = (y,w; imp-α2D14; +; +) Scale bar = 5 µm. (j) Loss of either Importin-β11 or Importin-α2 caused a threefold increase in ghost boutons. The frequency of ghost boutons could be returned to control levels by expression of the particular importin in the muscle, but not neurons. Also, muscle expression of NLS-Fz2-C rescued the importin-β11 phenotype but a control NLS-tagged GFP transgene did not. Ghost boutons were quantified; genotypes (as indicated by colors) and specific values are as follows: +/+ (y,w; FRT42D; +; +) = 1.0 ± 0.18 ghost boutons / NMJ, n = 10 animals, 59 NMJs; β11 −/− (y,w; imp-β1170 / Df; +; +) = 3.1 ± 0.4 n = 13 animals, 78 NMJs; α2 −/− (y,w; imp-α2D14; +; +) = 3.1 ± 0.4, n = 11 animals, 65 NMJs, p vs. imp-β1170 / Df >0.8; wit −/− (y,w; +; witA12/B11; +) = 0.71 ± 0.14, n = 11 animals, 65 NMJs, p vs. +/+ > 0.2; β11 −/− Neuron β11 (y,w; imp-β1170 / Df; elav-GAL4 / UAS-importin-β11-eGFP; +) = 3.2 ± 0.39 ghost boutons per NMJ, n = 8 animals, 48 NMJs, p vs. β11 −/− > 0.8; β11 −/− Muscle β11 (y,w; imp-β1170 / Df; 24B–GAL4 / UAS- importin-β11-eGFP; +) = 1.1 ± 0.17, n = 11 animals, 65 NMJs, p vs. +/+ > 0.5, vs. imp-β1170 / Df < 0.0001; α2 −/− Neuron α2 (y,w; imp-α2D14; elav-GAL4 / UAS-importin-α2; +) = 3.2 ± 0.33, n = 10 animals, 57 NMJs, p vs. α2−/− > 0.4; α2 −/− Muscle α2 (y,w; imp-α2D14; 24B–GAL4 / UAS-importin-α2; +) = 1.0 ± 0.19, n = 9 animals, 52 NMJs, p vs. +/+ > 0.8, vs. imp-α2D14 < 0.0001; β11 −/− Muscle Ketel = y,w; imp-β1170 / Df; 24B–GAL4 / UAS-Ketel; + (3.1 ± 0.38, n = 7 animals, 40 NMJs, p vs. β11 −/− >0.7; β11 −/− Muscle NLS-Fz2-C (w, UAS-myc-NLS-DFz2-C / + or Y; imp-β1170 / Df; 24B–GAL4 / +; +) = 1.1 ± 0.16 ghost boutons per NMJ, n = 14 animals, 78 NMJs, p vs. +/+ > 0.7, vs. imp-β1170 / Df < 0.0001; β11 −/− Muscle NLS-GFP (y,w; imp-β1170 / Df; UAS-NLS-GFP / 24B–GAL4; +) = 3.0 ± 0.48, n = 8 animals, 45 NMJs, p vs. imp-β1170 / Df > 0.4. *** p < 0.0001 vs. + / +.
Mentions: To test directly for an effect of the wg pathway and specifically Fz2-C nuclear import, we immunostained wg and dfz2 larvae with anti-HRP and anti-DLG (Supplementary Fig. 5) and counted instances at muscles 6 and 7 where boutons lacked surrounding DLG. In wild-type, ghost boutons were occasionally seen (Figure 6j) and found to lack apposite glutamate receptors (by GluRIIC staining) and the active zone protein Bruchpilot, but contained Synaptotagmin I immunoreactivity. When Wg / Fz2 signalling was perturbed, ghost bouton frequency increased: in hypomorphic wg1 mutants, their frequency doubled while in the more severe wgTS mutant, when shifted to a non-permissive temperature, their frequency was increased 4-fold (Supplementary Fig. 5). Similarly, in dfz2 mutants, ghost boutons increased nearly 3-fold (Supplementary Fig. 5). Neuronal overexpression of Wingless (Fig. 2)10 and hyperactivity mutations like eag1ShKS133 double mutants12 increase Wg signaling and nuclear Fz2-C. In these genotypes, ghost boutons were significantly fewer than in wild-type (Supplementary Fig. 5). Thus manipulations that decreased Wg / Fz2 signaling increased the frequency of ghost boutons and those that enhanced signaling diminished their occurrence.

Bottom Line: The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain.We found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large.Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway.

View Article: PubMed Central - PubMed

Affiliation: The F.M. Kirby Neurobiology Center, Children's Hospital Boston, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Synapse-to-nucleus signaling is critical for synaptic development and plasticity. In Drosophila, the ligand Wingless causes the C terminus of its Frizzled2 receptor (Fz2-C) to be cleaved and translocated from the postsynaptic density to nuclei. The mechanism of nuclear import is unknown and the developmental consequences of this translocation are uncertain. We found that Fz2-C localization to muscle nuclei required the nuclear import factors Importin-beta11 and Importin-alpha2 and that this pathway promoted the postsynaptic development of the subsynaptic reticulum (SSR), an elaboration of the postsynaptic plasma membrane. importin-beta11 (imp-beta11) and dfz2 mutants had less SSR, and some boutons lacked the postsynaptic marker Discs Large. These developmental defects in imp-beta11 mutants could be overcome by expression of Fz2-C fused to a nuclear localization sequence that can bypass Importin-beta11. Thus, Wnt-activated growth of the postsynaptic membrane is mediated by the synapse-to-nucleus translocation and active nuclear import of Fz2-C via a selective Importin-beta11/alpha2 pathway.

Show MeSH
Related in: MedlinePlus