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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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An NLS-tagged, truncated DFz2-C Construct Localizes to Synapses and is Imported Independently of Importin-β11(a) Organization of the full-length Frizzled2 protein and the truncated C-terminal construct. For this construct, the sequence after the cleavage site (arrowhead) was cloned behind a classical, monopartite nuclear localization signal and a myc epitope10. CRD = cysteine rich domain and TM = transmembrane domain. (b,c) Representative confocal images of third-instar larval NMJs of the genotype w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; + expressing myc-NLS-DFz2-C in the muscle and stained with an anti-myc (green) and anti-HRP (magenta). Myc immunoreactivity appears concentrated near boutons. Scale bar = 5 µm. (d–f) Representative single confocal slices through the equator of muscle nuclei (dashed circles) stained with anti-myc. (d) In the absence of the transgene (y,w; FRT42D; +; +), immunoreactivity is absent. (e,f) When myc-NLS-DFz2-C is expressed in wild-type (w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; +) or importin-β11 mutant larvae (w, UAS-myc-NLS-DFz2-C; imp-β1170 / Df; 24B–GAL4 / +;+) nuclear staining is observed. Scale bars = 10 µm.
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Figure 5: An NLS-tagged, truncated DFz2-C Construct Localizes to Synapses and is Imported Independently of Importin-β11(a) Organization of the full-length Frizzled2 protein and the truncated C-terminal construct. For this construct, the sequence after the cleavage site (arrowhead) was cloned behind a classical, monopartite nuclear localization signal and a myc epitope10. CRD = cysteine rich domain and TM = transmembrane domain. (b,c) Representative confocal images of third-instar larval NMJs of the genotype w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; + expressing myc-NLS-DFz2-C in the muscle and stained with an anti-myc (green) and anti-HRP (magenta). Myc immunoreactivity appears concentrated near boutons. Scale bar = 5 µm. (d–f) Representative single confocal slices through the equator of muscle nuclei (dashed circles) stained with anti-myc. (d) In the absence of the transgene (y,w; FRT42D; +; +), immunoreactivity is absent. (e,f) When myc-NLS-DFz2-C is expressed in wild-type (w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; +) or importin-β11 mutant larvae (w, UAS-myc-NLS-DFz2-C; imp-β1170 / Df; 24B–GAL4 / +;+) nuclear staining is observed. Scale bars = 10 µm.

Mentions: Although Importin-β11 was required for normal Fz2-C import, we attempted to bypass that requirement. We expressed in larval muscles the C-terminal 88 amino acids of Fz2-C with a nuclear localization signal (NLS) and a myc epitope (Fig. 5a) on its N-terminus, so as not to interfere with the PDZ-binding domain10. Attaching an NLS to cargoes can allow for import by alternative routes such as Importin-β122. In addition to diffuse cytoplasmic myc immunoreactivity, as seen previously10, we observed enrichment of myc-NLS-Fz2-C around synaptic boutons (Fig. 5b,c) likely due to binding via the PDZ-binding domain. We also observed nuclear myc-immunoreactivity in both wild-type and imp-β11 mutants (Fig. 5d–f). The nuclear immunoreactivity was punctate but these puncta were smaller than endogenous Fz2-C puncta and most abundant near the nucleolus. Thus the NLS-fused Fz2-C (like NLS-GFP; Supplementary Fig. 3) enters the nucleus even in the absence of Importin-β11; this raised the possibility that myc-NLS-Fz2-C could functionally substitute for normal Fz2 signaling.


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

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

An NLS-tagged, truncated DFz2-C Construct Localizes to Synapses and is Imported Independently of Importin-β11(a) Organization of the full-length Frizzled2 protein and the truncated C-terminal construct. For this construct, the sequence after the cleavage site (arrowhead) was cloned behind a classical, monopartite nuclear localization signal and a myc epitope10. CRD = cysteine rich domain and TM = transmembrane domain. (b,c) Representative confocal images of third-instar larval NMJs of the genotype w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; + expressing myc-NLS-DFz2-C in the muscle and stained with an anti-myc (green) and anti-HRP (magenta). Myc immunoreactivity appears concentrated near boutons. Scale bar = 5 µm. (d–f) Representative single confocal slices through the equator of muscle nuclei (dashed circles) stained with anti-myc. (d) In the absence of the transgene (y,w; FRT42D; +; +), immunoreactivity is absent. (e,f) When myc-NLS-DFz2-C is expressed in wild-type (w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; +) or importin-β11 mutant larvae (w, UAS-myc-NLS-DFz2-C; imp-β1170 / Df; 24B–GAL4 / +;+) nuclear staining is observed. Scale bars = 10 µm.
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Figure 5: An NLS-tagged, truncated DFz2-C Construct Localizes to Synapses and is Imported Independently of Importin-β11(a) Organization of the full-length Frizzled2 protein and the truncated C-terminal construct. For this construct, the sequence after the cleavage site (arrowhead) was cloned behind a classical, monopartite nuclear localization signal and a myc epitope10. CRD = cysteine rich domain and TM = transmembrane domain. (b,c) Representative confocal images of third-instar larval NMJs of the genotype w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; + expressing myc-NLS-DFz2-C in the muscle and stained with an anti-myc (green) and anti-HRP (magenta). Myc immunoreactivity appears concentrated near boutons. Scale bar = 5 µm. (d–f) Representative single confocal slices through the equator of muscle nuclei (dashed circles) stained with anti-myc. (d) In the absence of the transgene (y,w; FRT42D; +; +), immunoreactivity is absent. (e,f) When myc-NLS-DFz2-C is expressed in wild-type (w, UAS-myc-NLS-DFz2-C; +; 24B–GAL4 / +; +) or importin-β11 mutant larvae (w, UAS-myc-NLS-DFz2-C; imp-β1170 / Df; 24B–GAL4 / +;+) nuclear staining is observed. Scale bars = 10 µm.
Mentions: Although Importin-β11 was required for normal Fz2-C import, we attempted to bypass that requirement. We expressed in larval muscles the C-terminal 88 amino acids of Fz2-C with a nuclear localization signal (NLS) and a myc epitope (Fig. 5a) on its N-terminus, so as not to interfere with the PDZ-binding domain10. Attaching an NLS to cargoes can allow for import by alternative routes such as Importin-β122. In addition to diffuse cytoplasmic myc immunoreactivity, as seen previously10, we observed enrichment of myc-NLS-Fz2-C around synaptic boutons (Fig. 5b,c) likely due to binding via the PDZ-binding domain. We also observed nuclear myc-immunoreactivity in both wild-type and imp-β11 mutants (Fig. 5d–f). The nuclear immunoreactivity was punctate but these puncta were smaller than endogenous Fz2-C puncta and most abundant near the nucleolus. Thus the NLS-fused Fz2-C (like NLS-GFP; Supplementary Fig. 3) enters the nucleus even in the absence of Importin-β11; this raised the possibility that myc-NLS-Fz2-C could functionally substitute for normal Fz2 signaling.

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