The E3 ligase Thin controls homeostatic plasticity through neurotransmitter release repression

  1. Martin Baccino-Calace
  2. Katharina Schmidt
  3. Martin Müller  Is a corresponding author
  1. Department of Molecular Life Sciences, University of Zurich, Switzerland
  2. Zurich Ph.D. Program in Molecular Life Sciences, Switzerland
  3. Neuroscience Center Zurich, University of Zurich/ETH Zurich, Switzerland
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
An electrophysiology-based genetic screen identifies thin as a synaptic homeostasis gene.

(A) The number of putative E3 ubiquitin ligase-encoding genes (E3) and protein kinase-encoding genes (PK) as a function of total protein-coding gene number of C. cerevisiae, D. melanogaster, and H. …

Figure 1—figure supplement 1
Generation and prioritization of the E3 ligase-encoding gene list.

(A) Flow chart describing the prioritization process of the E3 list. Generation: First, we used the Gene Ontology (GO) search of Flybase (Larkin et al., 2021) to identify genes annotated to encode …

Figure 1—figure supplement 2
Homology between Thin and TRIM family proteins.

(A) Schematic representation of the domain organization of Drosophila Thin isoforms and its closest human TRIM family homologs. thin encodes an E3 ligase with a N-terminal tripartite motif (TRIM), …

Figure 2 with 1 supplement
Homeostatic plasticity requires presynaptic thin.

(A) Representative excitatory postsynaptic currents (EPSCs) (individual sweeps and averages are shown in light colors and black, respectively), and mEPSCs (insets) of wild-type (WT) (gray), thinΔA

Figure 2—figure supplement 1
Sustained homeostasis is impaired in thin mutants.

(A) Representative excitatory postsynaptic currents (EPSCs) (individual sweeps and averages are shown in light colors and black, respectively), and mEPSCs (insets) of wild-type (WT) (gray), GluRIIASP…

Figure 2—figure supplement 1—source data 1

Related to Figure 2—figure supplement 1.

Sustained homeostasis is impaired in thin mutants.

https://cdn.elifesciences.org/articles/71437/elife-71437-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Slight alterations in neuromuscular junction (NMJ) morphology upon genetic thin manipulations.

(A) Maximum intensity projection of a wild-type (WT) (left) and thinΔA mutant NMJ (right) (muscle 6) stained against the Drosophila neuronal membrane marker anti-HRP (‘HRP’) and the active-zone …

Figure 3—figure supplement 1
Postsynaptic thin expression does not affect presynaptic homeostatic plasticity (PHP) or baseline synaptic transmission.

(A) Maximum intensity projection of a control (24B-Gal4/+, ‘muscle-Gal4’, left and 24B-Gal4>UAS thin, ‘muscle-Gal4>UAS-thin’, right) neuromuscular junction (NMJ) (muscle 6) stained against the Drosop…

Figure 3—figure supplement 1—source data 1

Related to Figure 3—figure supplement 1.

Postsynaptic thin expression does not affect presynaptic homeostatic plasticity (PHP) or baseline synaptic transmission.

https://cdn.elifesciences.org/articles/71437/elife-71437-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Thin negatively regulates release-ready vesicle number.

(A) Representative excitatory postsynaptic currents (EPSCs) (individual sweeps and averages are shown in light colors and black, respectively), and mEPSCs (insets) of controls (elavc155-Gal4>UAS-mChe…

Figure 4—figure supplement 1
Presynaptic thinRNAi expression blocks presynaptic homeostatic plasticity (PHP) and induces a slight increase in AZ number.

(A) Maximum intensity projection of a control neuromuscular junction (NMJ) (elavc155-Gal4>UAS-mCherryRNAi, ‘Ctrl.’, left) and after presynaptic thinRNAi expression (elavc155-Gal4>UAS-thinRNAi, ‘thinR…

Figure 4—figure supplement 1—source data 1

Related to Figure 4—figure supplement 1.

Presynaptic thinRNAi expression blocks presynaptic homeostatic plasticity (PHP) and induces a slight increase in AZ number.

https://cdn.elifesciences.org/articles/71437/elife-71437-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
Thin localizes in close proximity to Dysbindin.

(A) Confocal maximum intensity projection of a representative neuromuscular junction (NMJ) branch (muscle 6–7) after presynaptic coexpression (elavc155-Gal4) of venus-tagged Dysbindin (UAS-venus-Dysb…

Figure 5—figure supplement 1
Dysbindin and Synapsin distribute in the periphery of synaptic boutons, endogenous Thin localizes close to Brp, and presynaptic dysbindin overexpression does not affect neuromuscular junction (NMJ) morphology.

(A) Confocal maximum intensity projection of a representative NMJ branch (muscle 6–7) after presynaptic expression (elavc155-Gal4) of venus-tagged Dysbindin (UAS-venus-dysbindin, ‘Dysbvenus’) …

Figure 5—figure supplement 1—source data 1

Related to Figure 5—figure supplement 1F.

Dysbindin and Synapsin distribute in the periphery of synaptic boutons, endogenous Thin localizes close to Brp, and presynaptic dysbindin overexpression does not affect neuromuscular junction (NMJ) morphology.

https://cdn.elifesciences.org/articles/71437/elife-71437-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 2
Thin localizes in close proximity to Dysbindin and Thin degrades Dysbindin in Drosophila S2 cells.

(A) Confocal images (single planes) of Drosophila S2 cells stained with anti-Dysbindin (green) and anti-Thin (magenta) under control conditions (top) and after dysbindin overexpression (UAS-venus-Dys…

Figure 5—figure supplement 2—source data 1

Related to Figure 5—figure supplement 2.

Thin localizes in close proximity to Dysbindin and Thin degrades Dysbindin in Drosophila S2 cells.

https://cdn.elifesciences.org/articles/71437/elife-71437-fig5-figsupp2-data1-v2.xlsx
Thin represses release through dysbindin.

(A) Representative excitatory postsynaptic currents (EPSCs) (individual sweeps and averages are shown in light colors and black, respectively), and mEPSCs (insets) of wild-type (WT) (gray) and …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Drosophila melanogaster)thinΔALaBeau-DiMenna et al., 2012
Genetic reagent (Drosophila melanogaster)UAS-abbaLaBeau-DiMenna et al., 2012
Genetic reagent (Drosophila melanogaster)UAS-mCherry-thinThis studyStock is available upon request
Genetic reagent (Drosophila melanogaster)GluRIIASP16Petersen et al., 1997
Genetic reagent (Drosophila melanogaster)dysbindin1Dickman and Davis, 2009
Genetic reagent (Drosophila melanogaster)UAS-thinRNAiPerkins et al., 2015RRID:BDSC_42826
Genetic reagent (Drosophila melanogaster)UAS-mCherryRNAi (P{VALIUM20-mCherry}attP2)Bloomington Drosophila Stock CenterRRID:BDSC_35785
Genetic reagent (Drosophila melanogaster)UAS-venus-dysbindinDickman and Davis, 2009
Genetic reagent (Drosophila melanogaster)elavc155-Gal4Bloomington Drosophila Stock CenterRRID:BDSC_458
Genetic reagent (Drosophila melanogaster)24B-Gal4Bloomington Drosophila Stock CenterRRID:BDSC_1767
Antibodyanti-Bruchpilot (nc82) (mouse monoclonal)DSHB, University of Iowa, USARRID:AB_2314866(1:100)
Antibodyanti-GFP (rabbit polyclonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# G10362, RRID:AB_2536526IF: (1:500)
WB: (1:1000)
Antibodyanti-GFP (mouse mono clonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# A-11120, RRID:AB_221568(1:500)
Antibodyanti-DsRed (mouse monoclonal)Santa Cruz BiotechnologySanta Cruz Biotechnology Cat# sc-390909, RRID:AB_2801575(1:500)
Antibodyanti-SYNORF1 (Synapsin, 3C11) (mouse monoclonal)DSHB, University of Iowa, USARRID:AB_528479(1:250)
Antibodyanti-Thin (guinea pig polyclonal)LaBeau-DiMenna et al., 2012Larva: (1:200)
S2:
(1:400)
Antibodyanti-HRP Alexa-Fluor 647 (goat polyclonal)Jackson ImmunoResearch LabsJackson ImmunoResearch Labs Cat# 123-605-021, RRID:AB_2338967(1:200)
AntibodyAnti-HA (mouse monoclonal)BiolegendBioLegend Cat# 901533, RRID:AB_2801249(1:1000)
AntibodyAnti-BetaTubulin (mouse monoclonal)DSHB, University of Iowa, USADSHB Cat# E7, RRID:AB_528499(1:1000)
AntibodyGoat anti-Mouse IgG (H+L) Secondary Antibody, HRP (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# 31430, RRID:AB_228307(1:2000)
AntibodyGoat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# 32460, RRID:AB_1185567(1:2000)
AntibodyAlexa-Fluor anti-mouse 488 (goat polyclonal)Thermo Fisher ScientificThermo Fisher Scientific Cat# A32723, RRID:AB_2633275(1:500)
AntibodyAlexa Fluor anti-guinea pig 555
(goat polyclonal)
Thermo Fisher ScientificThermo Fisher Scientific Cat# A-21435 RRID:AB_2535856(1:400)
AntibodyAtto 594 conjugated anti-mouse (goat polyclonal)Sigma-AldrichSigma-Aldrich Cat# 76,085(1:100)
AntibodyAbberior STAR 635P (goat polyclonal)AbberiorAbberior Cat# ST635P-1002-500 UG, RRID:AB_2893229(1:100)
Chemical compound, drugBouin’s fixativeSigma-AldrichHT-10132
Chemical compound, drugEthanolMerckCAS# 64-17-5
Chemical compound, drugProLong Gold AntifadeThermo Fisher ScientificP36930
Chemical compound, drugPhilanthotoxin-433Santa Cruz BiotechnologyCat# sc-255421
Chemical compound, drugSchneider’s Drosophila mediumGibcoCat# 21720024
Chemical compound, drugFuGENE HDPromegaCat# E2311
Chemical compound, drugParaformaldehydeMerckHT501128
Chemical compound, drugNP-40Thermo Fisher Scientific85,125
Chemical compound, drugDeoxycholateSigma-AldrichD6750
Chemical compound, drugcOmpleteSigma-Aldrich11697498001
Chemical compound, drugECL Prime Western Blotting Detection ReagentGE HealthcareCat# 28980926
Cell line (D. melanogaster)Drosophila Schneider 2 (S2) CellsThermo Fisher ScientificCat# R69007
Commercial assay, kitNitrocellulose membraneAmersham Hibond GE HealthcareCat# 88,018
Recombinant DNA reagentpMT-Gal4AddgeneRRID:Addgene_53366
Software, algorithmFiji / ImageJhttps://fiji.scRRID:SCR_002285Version 1.51n
Software, algorithmClampexAxon CNS, Molecular DevicesRRID:SCR_011323
Software, algorithmLeica Application Suite XLeica MicrosystemsRRID:SCR_013673
Software, algorithmHuygens Softwarehttps://svi.nl/HuygensSoftwareRRID:SCR_014237
Software, algorithmIgor ProWaveMetricsRRID:SCR_000325Version 6.37
Software, algorithmNeuroMaticRothman and Silver, 2018RRID:SCR_004186Version 3.0c
Software, algorithmNumPyhttps://www.numpy.orgRRID:SCR_008633
Software, algorithmSciPyhttps://www.scipy.orgRRID:SCR_008058
Software, algorithmIPythonhttp://ipython.orgRRID:SCR_001658
Software, algorithmNeohttp://neuralensemble.org/neoRRID:SCR_000634
Software, algorithmShapely(Gillies, 2007) https://github.com/shapely/shapely
Software, algorithmRStudio(R Studio Team, 2020)

http://www.rstudio.com/
RRID:SCR_000432Version 2021.09.0
Software, algorithmpwr-package(Champely, 2020)

https://github.com/heliosdrm/pwr
Software, algorithmGNU Image Manipulation Programhttps://www.gimp.org/RRID:SCR_003182Version 2.8.10
Software, algorithmInkscapehttp://www.inkscape.orgRRID:SCR_014479Version 0.92.2
Software, algorithmAffinity Designerhttps://affinity.serif.com/en-us/designer/RRID:SCR_016952Version 1.10.4

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/71437/elife-71437-transrepform1-v2.pdf
Supplementary file 1

Summary table of electrophysiology data for the genetic screen.

Data are mean ± SEM. UAS-RNAis were driven in neurons by elavc155-Gal4, and elavc155-Gal4/Y served as the control. We tested 157 putative E3 ligase-encoding genes and 11 E3-associated genes, using 180 lines (UAS-RNAi or mutants; some genes were targeted with multiple lines; mean n = 4 NMJs per line, range 3–12 per line). Control data were continuously collected throughout the genetic screen. See Materials and methods for further details.

https://cdn.elifesciences.org/articles/71437/elife-71437-supp1-v2.xlsx

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