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Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

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The localization and levels of Gtaxin at the postsynaptic specialization are Akt1-dependent, and overexpression of a constitutively active form of Akt1 creates ectopic membranes distant from the synaptic region. Gtaxin localization and levels were examined in animals with reduced Akt1 function or muscle directed expression of a constitutively active form of Akt1, Akt1CA. All animals in this experiment also carried the muscle-specific driver Mef2-GAL4 and UAS-mCD8-GFP transgenes. Gtaxin and GluRIIA were detected with anti-Gtx (red) and anti-GluRIIA (grayscale) antibodies. Panels (A–C), (E–G), and (I–K) are each from a single animal. Panels (D), (H), and (L) are anti-GluRIIA staining each from a single larva. A–D: Control animals had no Akt1-bearing transgene either Akt1RNAi or Akt1CA (labeled as +). Gtaxin is concentrated at the SSR, colocalizing with mCD8-GFP (arrows). GluRIIA is also highly concentrated at the NMJ specialization. E–H: Animals expressing UAS-Akt1RNAi showed dramatic reductions in the levels of mCD8-GFP at the SSR (E) and loss of Gtaxin at the synapse (F), as well as mislocalization of GluRIIA (H). I–L: Overexpression of Akt1CA caused ectopic mCD8 patches throughout the muscle (I), as well as increased levels of Gtaxin (J). The normal distribution of Gtaxin at the SSR was lost, with mislocalized Gtaxin patches evident throughout the muscle cell (J). Correct GluRIIA localization was maintained in these animals (L). M–O: Transmission electron microscope photomicrographs show ectopic membranous structures in muscles overexpressing the constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). Arrows point to infoldings of multilayered membranes in the cytosol or underneath the plasma membrane. Scale bar in (A–L), 50 µm, in (M–O), 0.1 µm.
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fig06: The localization and levels of Gtaxin at the postsynaptic specialization are Akt1-dependent, and overexpression of a constitutively active form of Akt1 creates ectopic membranes distant from the synaptic region. Gtaxin localization and levels were examined in animals with reduced Akt1 function or muscle directed expression of a constitutively active form of Akt1, Akt1CA. All animals in this experiment also carried the muscle-specific driver Mef2-GAL4 and UAS-mCD8-GFP transgenes. Gtaxin and GluRIIA were detected with anti-Gtx (red) and anti-GluRIIA (grayscale) antibodies. Panels (A–C), (E–G), and (I–K) are each from a single animal. Panels (D), (H), and (L) are anti-GluRIIA staining each from a single larva. A–D: Control animals had no Akt1-bearing transgene either Akt1RNAi or Akt1CA (labeled as +). Gtaxin is concentrated at the SSR, colocalizing with mCD8-GFP (arrows). GluRIIA is also highly concentrated at the NMJ specialization. E–H: Animals expressing UAS-Akt1RNAi showed dramatic reductions in the levels of mCD8-GFP at the SSR (E) and loss of Gtaxin at the synapse (F), as well as mislocalization of GluRIIA (H). I–L: Overexpression of Akt1CA caused ectopic mCD8 patches throughout the muscle (I), as well as increased levels of Gtaxin (J). The normal distribution of Gtaxin at the SSR was lost, with mislocalized Gtaxin patches evident throughout the muscle cell (J). Correct GluRIIA localization was maintained in these animals (L). M–O: Transmission electron microscope photomicrographs show ectopic membranous structures in muscles overexpressing the constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). Arrows point to infoldings of multilayered membranes in the cytosol or underneath the plasma membrane. Scale bar in (A–L), 50 µm, in (M–O), 0.1 µm.

Mentions: Based on the similar ultrastructural changes in the SSR and muscle membrane organization resulting from reductions in Gtaxin and Akt1 function, Gtaxin was a logical candidate as a downstream target of Akt1 activity. To explore this possibility, we examined Gtaxin levels and distribution in animals with muscle-specific expression of Akt1RNAi (Mef2-GAL4>UAS-Akt1RNAi) or a constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). In wild-type animals, Gtaxin immunoreactivity is concentrated at the SSR [Fig. 6(B,C)], and muscle-directed RNAi of Akt1 greatly reduced Gtaxin levels at this postsynaptic specialization [Fig. 6(F,G)]. Gtaxin has been implicated in SSR formation not only on account of the reduction of SSR complexity in Gtaxin mutant but also from the production of ectopic, mCD8-GFP labeled membranous structures in animals overexpressing wild-type Gtaxin (Gorczyca et al., 2007). Muscle-directed expression of Akt1CA produced membranous structures with the same visible features. In these animals, Gtaxin was present at increased levels and localized to patches throughout the muscle [Fig. 6(I–K)]. These ectopic membrane elaborations were confirmed at the TEM level and are structurally similar to those documented in animals overexpressing Gtaxin in the muscle [Fig. 6(M–O)].


Akt regulates glutamate receptor trafficking and postsynaptic membrane elaboration at the Drosophila neuromuscular junction.

Lee HG, Zhao N, Campion BK, Nguyen MM, Selleck SB - Dev Neurobiol (2013)

The localization and levels of Gtaxin at the postsynaptic specialization are Akt1-dependent, and overexpression of a constitutively active form of Akt1 creates ectopic membranes distant from the synaptic region. Gtaxin localization and levels were examined in animals with reduced Akt1 function or muscle directed expression of a constitutively active form of Akt1, Akt1CA. All animals in this experiment also carried the muscle-specific driver Mef2-GAL4 and UAS-mCD8-GFP transgenes. Gtaxin and GluRIIA were detected with anti-Gtx (red) and anti-GluRIIA (grayscale) antibodies. Panels (A–C), (E–G), and (I–K) are each from a single animal. Panels (D), (H), and (L) are anti-GluRIIA staining each from a single larva. A–D: Control animals had no Akt1-bearing transgene either Akt1RNAi or Akt1CA (labeled as +). Gtaxin is concentrated at the SSR, colocalizing with mCD8-GFP (arrows). GluRIIA is also highly concentrated at the NMJ specialization. E–H: Animals expressing UAS-Akt1RNAi showed dramatic reductions in the levels of mCD8-GFP at the SSR (E) and loss of Gtaxin at the synapse (F), as well as mislocalization of GluRIIA (H). I–L: Overexpression of Akt1CA caused ectopic mCD8 patches throughout the muscle (I), as well as increased levels of Gtaxin (J). The normal distribution of Gtaxin at the SSR was lost, with mislocalized Gtaxin patches evident throughout the muscle cell (J). Correct GluRIIA localization was maintained in these animals (L). M–O: Transmission electron microscope photomicrographs show ectopic membranous structures in muscles overexpressing the constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). Arrows point to infoldings of multilayered membranes in the cytosol or underneath the plasma membrane. Scale bar in (A–L), 50 µm, in (M–O), 0.1 µm.
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fig06: The localization and levels of Gtaxin at the postsynaptic specialization are Akt1-dependent, and overexpression of a constitutively active form of Akt1 creates ectopic membranes distant from the synaptic region. Gtaxin localization and levels were examined in animals with reduced Akt1 function or muscle directed expression of a constitutively active form of Akt1, Akt1CA. All animals in this experiment also carried the muscle-specific driver Mef2-GAL4 and UAS-mCD8-GFP transgenes. Gtaxin and GluRIIA were detected with anti-Gtx (red) and anti-GluRIIA (grayscale) antibodies. Panels (A–C), (E–G), and (I–K) are each from a single animal. Panels (D), (H), and (L) are anti-GluRIIA staining each from a single larva. A–D: Control animals had no Akt1-bearing transgene either Akt1RNAi or Akt1CA (labeled as +). Gtaxin is concentrated at the SSR, colocalizing with mCD8-GFP (arrows). GluRIIA is also highly concentrated at the NMJ specialization. E–H: Animals expressing UAS-Akt1RNAi showed dramatic reductions in the levels of mCD8-GFP at the SSR (E) and loss of Gtaxin at the synapse (F), as well as mislocalization of GluRIIA (H). I–L: Overexpression of Akt1CA caused ectopic mCD8 patches throughout the muscle (I), as well as increased levels of Gtaxin (J). The normal distribution of Gtaxin at the SSR was lost, with mislocalized Gtaxin patches evident throughout the muscle cell (J). Correct GluRIIA localization was maintained in these animals (L). M–O: Transmission electron microscope photomicrographs show ectopic membranous structures in muscles overexpressing the constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). Arrows point to infoldings of multilayered membranes in the cytosol or underneath the plasma membrane. Scale bar in (A–L), 50 µm, in (M–O), 0.1 µm.
Mentions: Based on the similar ultrastructural changes in the SSR and muscle membrane organization resulting from reductions in Gtaxin and Akt1 function, Gtaxin was a logical candidate as a downstream target of Akt1 activity. To explore this possibility, we examined Gtaxin levels and distribution in animals with muscle-specific expression of Akt1RNAi (Mef2-GAL4>UAS-Akt1RNAi) or a constitutively active form of Akt1 (Mef2-GAL4>UAS-Akt1CA). In wild-type animals, Gtaxin immunoreactivity is concentrated at the SSR [Fig. 6(B,C)], and muscle-directed RNAi of Akt1 greatly reduced Gtaxin levels at this postsynaptic specialization [Fig. 6(F,G)]. Gtaxin has been implicated in SSR formation not only on account of the reduction of SSR complexity in Gtaxin mutant but also from the production of ectopic, mCD8-GFP labeled membranous structures in animals overexpressing wild-type Gtaxin (Gorczyca et al., 2007). Muscle-directed expression of Akt1CA produced membranous structures with the same visible features. In these animals, Gtaxin was present at increased levels and localized to patches throughout the muscle [Fig. 6(I–K)]. These ectopic membrane elaborations were confirmed at the TEM level and are structurally similar to those documented in animals overexpressing Gtaxin in the muscle [Fig. 6(M–O)].

Bottom Line: The single Drosophila Akt family member, Akt1 selectively altered the postsynaptic targeting of one glutamate receptor subunit, GluRIIA, and was required for the expansion of a specialized postsynaptic membrane compartment, the subsynaptic reticulum (SSR).Several lines of evidence indicated that Akt1 influences SSR assembly by regulation of Gtaxin, a Drosophila t-SNARE protein (Gorczyca et al., 2007) in a manner independent of the mislocalization of GluRIIA.Our findings show that Akt1 governs two critical elements of synapse development, neurotransmitter receptor localization, and postsynaptic membrane elaboration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, 16802.

Show MeSH
Related in: MedlinePlus