Limits...
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.

Show MeSH

Related in: MedlinePlus

Loss of Nuclear DFz2-C Leads to Reduced SSR Thickness at the EM Level(a–c) Representative electron micrographs of type Ib boutons on muscles 6 and 7 in segment A2 of wild-type (y,w; FRT42D; +; +), importin-β11 mutants (y,w; imp-β1170 / Df; +; +) and dfz2 mutants (y,w; +; dfz2C1 / Df(3L)ED4782; +). Both importin-β11 and dfz2 mutants display regions of subsynaptic reticulum (SSR, false colored in red) that are thinner than wild-type controls. In all images, scale bar = 500 nm. (d) Scatterplot quantification of the thickness of the SSR in wild-type (blue circles, y,w; FRT42D; +; +)dfz2 mutants (crimson triangles, y,w; +; dfz2C1 / Df(3L)ED4782; +), importin-β11 mutants (green squares, y,w; imp-β1170 / Df; +; +), and importin-β11 mutants expressing NLS-Fz2-C in the muscle (green diamonds, w, UAS-myc-NLS-Fz2-C; imp-β1170 / Df; 24B–GAL4 / +). Each symbol represents the average thickness around a single bouton. Restoration of nuclear Fz2-C in the importin-β11 mutant rescues SSR thickness to nearly wild-type levels. Specific values are as follows: +/+ = 956.4 ± 38.66 nm, n = 5 animals, 52 boutons; β11 −/− (y,w; imp-β1170 / Df; +; +) = 558.5 ± 33.74 nm, n = 5 animals, 17 boutons; dfz2 −/− (y,w; +; dfz2C1 / Df(3L)ED4782; +) = 567.1 ± 41.71 nm, n = 3 animals, 26 boutons, p vs. imp-β1170 / Df > 0.8; β11 −/− Muscle NLS-Fz2-C = 841.3 ± 28.72 nm, n = 5 animals, 103 boutons, p vs. +/+ > 0.2, vs. imp-β1170 / Df < 0.0001. *** p < 0.0001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2913881&req=5

Figure 7: Loss of Nuclear DFz2-C Leads to Reduced SSR Thickness at the EM Level(a–c) Representative electron micrographs of type Ib boutons on muscles 6 and 7 in segment A2 of wild-type (y,w; FRT42D; +; +), importin-β11 mutants (y,w; imp-β1170 / Df; +; +) and dfz2 mutants (y,w; +; dfz2C1 / Df(3L)ED4782; +). Both importin-β11 and dfz2 mutants display regions of subsynaptic reticulum (SSR, false colored in red) that are thinner than wild-type controls. In all images, scale bar = 500 nm. (d) Scatterplot quantification of the thickness of the SSR in wild-type (blue circles, y,w; FRT42D; +; +)dfz2 mutants (crimson triangles, y,w; +; dfz2C1 / Df(3L)ED4782; +), importin-β11 mutants (green squares, y,w; imp-β1170 / Df; +; +), and importin-β11 mutants expressing NLS-Fz2-C in the muscle (green diamonds, w, UAS-myc-NLS-Fz2-C; imp-β1170 / Df; 24B–GAL4 / +). Each symbol represents the average thickness around a single bouton. Restoration of nuclear Fz2-C in the importin-β11 mutant rescues SSR thickness to nearly wild-type levels. Specific values are as follows: +/+ = 956.4 ± 38.66 nm, n = 5 animals, 52 boutons; β11 −/− (y,w; imp-β1170 / Df; +; +) = 558.5 ± 33.74 nm, n = 5 animals, 17 boutons; dfz2 −/− (y,w; +; dfz2C1 / Df(3L)ED4782; +) = 567.1 ± 41.71 nm, n = 3 animals, 26 boutons, p vs. imp-β1170 / Df > 0.8; β11 −/− Muscle NLS-Fz2-C = 841.3 ± 28.72 nm, n = 5 animals, 103 boutons, p vs. +/+ > 0.2, vs. imp-β1170 / Df < 0.0001. *** p < 0.0001.

Mentions: Presynaptic boutons in imp-β11 mutants were ultrastructurally normal with regard to many parameters (Table 1). However, the SSR surrounding imp-β11 and dfz2 boutons (Fig. 7a–c) was only 58% as thick as in controls (Fig. 7d). Similar reductions were seen in SSR area and the ratio of SSR area to bouton area, but not bouton area itself (Supplementary Fig. 6a–c). The density, though, of SSR folds was unchanged (Table 1). Moreover, the phenotypes of imp-β11 and dfz2 were statistically indistinguishable. Two of 26 dfz2 boutons lacked any surrounding SSR (Supplementary Fig. 7b), potentially an ultrastructural correlate of ghost boutons and similar to those observed in wgTS larvae14. Although no imp-β11 boutons were seen that completely lacked SSR, many had very little and might have been scored as ghost boutons by immunocytochemistry (Supplementary Fig. 7a). This loss of SSR was not due to a general defect in muscle growth or membrane traffic. Muscle size, input resistance and miniature EPSP amplitude in imp-β11 larvae were all unchanged16. Moreover, despite thinner SSR, glutamate receptor levels were normal (Supplementary Fig. S8) and receptors correctly apposed active zones16 everywhere except the ghost boutons. Thus, the SSR reduction is not an indication of broadly compromised muscles.


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

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

Loss of Nuclear DFz2-C Leads to Reduced SSR Thickness at the EM Level(a–c) Representative electron micrographs of type Ib boutons on muscles 6 and 7 in segment A2 of wild-type (y,w; FRT42D; +; +), importin-β11 mutants (y,w; imp-β1170 / Df; +; +) and dfz2 mutants (y,w; +; dfz2C1 / Df(3L)ED4782; +). Both importin-β11 and dfz2 mutants display regions of subsynaptic reticulum (SSR, false colored in red) that are thinner than wild-type controls. In all images, scale bar = 500 nm. (d) Scatterplot quantification of the thickness of the SSR in wild-type (blue circles, y,w; FRT42D; +; +)dfz2 mutants (crimson triangles, y,w; +; dfz2C1 / Df(3L)ED4782; +), importin-β11 mutants (green squares, y,w; imp-β1170 / Df; +; +), and importin-β11 mutants expressing NLS-Fz2-C in the muscle (green diamonds, w, UAS-myc-NLS-Fz2-C; imp-β1170 / Df; 24B–GAL4 / +). Each symbol represents the average thickness around a single bouton. Restoration of nuclear Fz2-C in the importin-β11 mutant rescues SSR thickness to nearly wild-type levels. Specific values are as follows: +/+ = 956.4 ± 38.66 nm, n = 5 animals, 52 boutons; β11 −/− (y,w; imp-β1170 / Df; +; +) = 558.5 ± 33.74 nm, n = 5 animals, 17 boutons; dfz2 −/− (y,w; +; dfz2C1 / Df(3L)ED4782; +) = 567.1 ± 41.71 nm, n = 3 animals, 26 boutons, p vs. imp-β1170 / Df > 0.8; β11 −/− Muscle NLS-Fz2-C = 841.3 ± 28.72 nm, n = 5 animals, 103 boutons, p vs. +/+ > 0.2, vs. imp-β1170 / Df < 0.0001. *** p < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2913881&req=5

Figure 7: Loss of Nuclear DFz2-C Leads to Reduced SSR Thickness at the EM Level(a–c) Representative electron micrographs of type Ib boutons on muscles 6 and 7 in segment A2 of wild-type (y,w; FRT42D; +; +), importin-β11 mutants (y,w; imp-β1170 / Df; +; +) and dfz2 mutants (y,w; +; dfz2C1 / Df(3L)ED4782; +). Both importin-β11 and dfz2 mutants display regions of subsynaptic reticulum (SSR, false colored in red) that are thinner than wild-type controls. In all images, scale bar = 500 nm. (d) Scatterplot quantification of the thickness of the SSR in wild-type (blue circles, y,w; FRT42D; +; +)dfz2 mutants (crimson triangles, y,w; +; dfz2C1 / Df(3L)ED4782; +), importin-β11 mutants (green squares, y,w; imp-β1170 / Df; +; +), and importin-β11 mutants expressing NLS-Fz2-C in the muscle (green diamonds, w, UAS-myc-NLS-Fz2-C; imp-β1170 / Df; 24B–GAL4 / +). Each symbol represents the average thickness around a single bouton. Restoration of nuclear Fz2-C in the importin-β11 mutant rescues SSR thickness to nearly wild-type levels. Specific values are as follows: +/+ = 956.4 ± 38.66 nm, n = 5 animals, 52 boutons; β11 −/− (y,w; imp-β1170 / Df; +; +) = 558.5 ± 33.74 nm, n = 5 animals, 17 boutons; dfz2 −/− (y,w; +; dfz2C1 / Df(3L)ED4782; +) = 567.1 ± 41.71 nm, n = 3 animals, 26 boutons, p vs. imp-β1170 / Df > 0.8; β11 −/− Muscle NLS-Fz2-C = 841.3 ± 28.72 nm, n = 5 animals, 103 boutons, p vs. +/+ > 0.2, vs. imp-β1170 / Df < 0.0001. *** p < 0.0001.
Mentions: Presynaptic boutons in imp-β11 mutants were ultrastructurally normal with regard to many parameters (Table 1). However, the SSR surrounding imp-β11 and dfz2 boutons (Fig. 7a–c) was only 58% as thick as in controls (Fig. 7d). Similar reductions were seen in SSR area and the ratio of SSR area to bouton area, but not bouton area itself (Supplementary Fig. 6a–c). The density, though, of SSR folds was unchanged (Table 1). Moreover, the phenotypes of imp-β11 and dfz2 were statistically indistinguishable. Two of 26 dfz2 boutons lacked any surrounding SSR (Supplementary Fig. 7b), potentially an ultrastructural correlate of ghost boutons and similar to those observed in wgTS larvae14. Although no imp-β11 boutons were seen that completely lacked SSR, many had very little and might have been scored as ghost boutons by immunocytochemistry (Supplementary Fig. 7a). This loss of SSR was not due to a general defect in muscle growth or membrane traffic. Muscle size, input resistance and miniature EPSP amplitude in imp-β11 larvae were all unchanged16. Moreover, despite thinner SSR, glutamate receptor levels were normal (Supplementary Fig. S8) and receptors correctly apposed active zones16 everywhere except the ghost boutons. Thus, the SSR reduction is not an indication of broadly compromised muscles.

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