Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan:70:11-21.
doi: 10.1016/j.mcn.2015.11.004. Epub 2015 Nov 17.

The Notch ligand E3 ligase, Mind Bomb1, regulates glutamate receptor localization in Drosophila

Morgan Sturgeon  1 , Dustin Davis  1 , Amanda Albers  1 , Derek Beatty  1 , Rik Austin  1 , Matt Ferguson  1 , Brittany Tounsel  1 , Faith L W Liebl  2
Affiliations

The Notch ligand E3 ligase, Mind Bomb1, regulates glutamate receptor localization in Drosophila

Morgan Sturgeon et al. Mol Cell Neurosci. 2016 Jan.

Abstract

The postsynaptic density (PSD) is a protein-rich network important for the localization of postsynaptic glutamate receptors (GluRs) and for signaling downstream of these receptors. Although hundreds of PSD proteins have been identified, many are functionally uncharacterized. We conducted a reverse genetic screen for mutations that affected GluR localization using Drosophila genes that encode homologs of mammalian PSD proteins. 42.8% of the mutants analyzed exhibited a significant change in GluR localization at the third instar larval neuromuscular junction (NMJ), a model synapse that expresses homologs of AMPA receptors. We identified the E3 ubiquitin ligase, Mib1, which promotes Notch signaling, as a regulator of synaptic GluR localization. Mib1 positively regulates the localization of the GluR subunits GluRIIA, GluRIIB, and GluRIIC. Mutations in mib1 and ubiquitous expression of Mib1 that lacks its ubiquitin ligase activity result in the loss of synaptic GluRIIA-containing receptors. In contrast, overexpression of Mib1 in all tissues increases postsynaptic levels of GluRIIA. Cellular levels of Mib1 are also important for the structure of the presynaptic motor neuron. While deficient Mib1 signaling leads to overgrowth of the NMJ, ubiquitous overexpression of Mib1 results in a reduction in the number of presynaptic motor neuron boutons and branches. These synaptic changes may be secondary to attenuated glutamate release from the presynaptic motor neuron in mib1 mutants as mib1 mutants exhibit significant reductions in the vesicle-associated protein cysteine string protein and in the frequency of spontaneous neurotransmission.

Keywords: Drosophila neuromuscular junction; Glutamate receptors; Postsynaptic density; Synapse; Synaptic transmission.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Mutations in Drosophila genes encoding homologs of mammalian PSD proteins lead to a reduction in GluRIIA cluster sizes
(A) Confocal images of third instar larval 6/7 NMJs immunolabeled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label postsynaptic glutamate receptor clusters containing the GluRIIA subunit. Inset panels show high resolution terminal boutons. Scale bar = 20 µm. (B) Histogram showing GluRIIA cluster sizes for genotypes represented in A. (C) Quantification of the number of boutons (left) and branches (right) indicative of presynaptic motor neuron morphology.
Figure 2
Figure 2. Mutations in Drosophila genes encoding homologs of mammalian PSD proteins lead to an increase in GluRIIA cluster sizes
(A) Representative confocal micrographs of 6/7 NMJs immunolabeled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label GluRIIA-containing glutamate receptor clusters. Inset panels show high resolution terminal boutons. Scale bar = 20 µm. (B) Quantification of GluRIIA cluster sizes for genotypes shown in A. (C) Quantification of characteristics representative of presynaptic motor neuron morphology including the number of boutons (left) and branches (right).
Figure 3
Figure 3. Mib1 is important for the clustering of GluRIIA-containing receptors
(A) Control and mib1 mutant confocal images showing representative 6/7 NMJs from third instar larvae. Preparations were immunolabled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label GluRIIA-containing glutamate receptor clusters. Inset panels show high resolution terminal boutons. Scale bar = 20 µm. (B) Histograms showing quantification of GluRIIA cluster sizes (left) and GluRIIA relative fluorescence intensity (right) for genotypes shown in A. (C) Quantification of characteristics representative of presynaptic motor neuron morphology including the number of boutons (top) and branches (bottom).
Figure 4
Figure 4. Mib1 positively regulates synaptic levels of GluRIIB and GluRIIC but does not affect glur transcript levels
High resolution confocal micrographs showing third instar larvae terminal boutons of the 6/7 NMJ immunolabeled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label GluRIIB-containing glutamate receptor clusters (A) or GluRIIC-containing glutamate receptor clusters (C). Scale bar = 5 µm. (B) Quantification of GluRIIB cluster sizes (left) and relative fluorescence intensities (right) for genotypes shown in A. (D) Quantification of GluRIIC cluster sizes (left) and relative GluRIIC fluorescence intensity (right) for genotypes shown in B. (E) Quantification qRT-PCR ΔΔC(t) values normalized to the control, w1118, using gene-specific primers for glurIIA (left), glurIIB (middle), and glurIIC (right).
Figure 5
Figure 5. Expression of Mib1 lacking the RING finger domains leads to the loss of synaptic GluRIIA
A mib1 transgene lacking the region encoding the three C-terminal RING finger domains, UAS-mib1Δ3RF, was expressed in all tissues using the Actin5c-Gal4 driver. (A) Representative third instar larvae 6/7 NMJs immunolabeled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label postsynaptic glutamate receptor clusters containing the GluRIIA subunit. Inset panels show high resolution terminal boutons. Scale bar = 20 µm. (B) Quantification of GluRIIA cluster sizes (left) and GluRIIA fluorescence intensities (right) for genotypes represented in A. (C) Characterization of presynaptic motor neuron morphology by quantification of the number of boutons (top) and branches (bottom).
Figure 6
Figure 6. Overexpression of Mib1 leads to an increase in synaptic GluRIIA and a reduction in the size of the presynaptic motor neuron
Overexpression of mib1 was achieved by expressing UAS-mib1wt in all tissues using the Actin5c-Gal4 driver. (A) Confocal micrographs showing 6/7 NMJs immunolabeled with α-HRP to label presynaptic motor neurons (magenta) and α-GluRIIA (green) to label postsynaptic GluRIIA-containing glutamate receptor clusters. Inset panels show high resolution terminal boutons. Scale bar = 20 µm. (B) Histograms showing quantification of GluRIIA cluster sizes (left) and GluRIIA fluorescence intensities (right) for genotypes represented in A. (C) Histograms showing quantification of the number of boutons (top) and branches (bottom).
Figure 7
Figure 7. Mib1 is important for the localization of CSP and FasII at the synapse
High resolution confocal micrographs showing terminal boutons of 6/7 NMJs from third instar larvae immunolabeled with α-HRP (magenta) and α-Brp (green, A), α-CSP (green, B), α-DLG (green, C), or α-FasII (green, D). Scale bar = 5 µm. Right histograms show quantification of mean normalized fluorescence intensities.
Figure 8
Figure 8. Mib1 negatively regulates spontaneous synaptic transmission
Spontaneous (mEJCs) and evoked junctional currents (eEJCs) were recorded from third instar larvae after voltage clamp of muscle 6 at −60 mV. (A) Representative mEJCs from control and mib1 mutants. (B) Quantification of mEJC frequency and amplitudes (top) and eEJC amplitudes and quantal content (bottom).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Alie A, Manuel M. The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans. BMC Evol Biol. 2010;10:34. - PMC - PubMed
    1. Anggono V, Huganir RL. Regulation of AMPA receptor trafficking and synaptic plasticity. Current opinion in neurobiology. 2012;22:461–469. - PMC - PubMed
    1. Augustin H, Grosjean Y, Chen K, Sheng Q, Featherstone DE. Nonvesicular release of glutamate by glial xCT transporters suppresses glutamate receptor clustering in vivo. J Neurosci. 2007;27:111–123. - PMC - PubMed
    1. Bangash MA, Park JM, Melnikova T, Wang D, Jeon SK, Lee D, Syeda S, Kim J, Kouser M, Schwartz J, Cui Y, Zhao X, Speed HE, Kee SE, Tu JC, Hu JH, Petralia RS, Linden DJ, Powell CM, Savonenko A, Xiao B, Worley PF. Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism. Cell. 2011;145:758–772. - PMC - PubMed
    1. Bayes A, van de Lagemaat LN, Collins MO, Croning MD, Whittle IR, Choudhary JS, Grant SG. Characterization of the proteome, diseases and evolution of the human postsynaptic density. Nat Neurosci. 2011;14:19–21. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources