(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. …
(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 …
(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), …
(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 …
Related to Figure 2.
(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…
Related to Figure 2—figure supplement 1.
Sustained homeostasis is impaired in thin mutants.
(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 …
Related to Figure 3.
(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…
Related to Figure 3—figure supplement 1.
Postsynaptic thin expression does not affect presynaptic homeostatic plasticity (PHP) or baseline synaptic transmission.
(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…
Related to Figure 4.
(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…
Related to Figure 4—figure supplement 1.
Presynaptic thinRNAi expression blocks presynaptic homeostatic plasticity (PHP) and induces a slight increase in AZ number.
(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…
Related to Figure 5.
(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’) …
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.
(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…
Related to Figure 5—figure supplement 2.
Thin localizes in close proximity to Dysbindin and Thin degrades Dysbindin in Drosophila S2 cells.
(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 …
Related to Figure 6.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (Drosophila melanogaster) | thinΔA | LaBeau-DiMenna et al., 2012 | ||
Genetic reagent (Drosophila melanogaster) | UAS-abba | LaBeau-DiMenna et al., 2012 | ||
Genetic reagent (Drosophila melanogaster) | UAS-mCherry-thin | This study | Stock is available upon request | |
Genetic reagent (Drosophila melanogaster) | GluRIIASP16 | Petersen et al., 1997 | ||
Genetic reagent (Drosophila melanogaster) | dysbindin1 | Dickman and Davis, 2009 | ||
Genetic reagent (Drosophila melanogaster) | UAS-thinRNAi | Perkins et al., 2015 | RRID:BDSC_42826 | |
Genetic reagent (Drosophila melanogaster) | UAS-mCherryRNAi (P{VALIUM20-mCherry}attP2) | Bloomington Drosophila Stock Center | RRID:BDSC_35785 | |
Genetic reagent (Drosophila melanogaster) | UAS-venus-dysbindin | Dickman and Davis, 2009 | ||
Genetic reagent (Drosophila melanogaster) | elavc155-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_458 | |
Genetic reagent (Drosophila melanogaster) | 24B-Gal4 | Bloomington Drosophila Stock Center | RRID:BDSC_1767 | |
Antibody | anti-Bruchpilot (nc82) (mouse monoclonal) | DSHB, University of Iowa, USA | RRID:AB_2314866 | (1:100) |
Antibody | anti-GFP (rabbit polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526 | IF: (1:500) WB: (1:1000) |
Antibody | anti-GFP (mouse mono clonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A-11120, RRID:AB_221568 | (1:500) |
Antibody | anti-DsRed (mouse monoclonal) | Santa Cruz Biotechnology | Santa Cruz Biotechnology Cat# sc-390909, RRID:AB_2801575 | (1:500) |
Antibody | anti-SYNORF1 (Synapsin, 3C11) (mouse monoclonal) | DSHB, University of Iowa, USA | RRID:AB_528479 | (1:250) |
Antibody | anti-Thin (guinea pig polyclonal) | LaBeau-DiMenna et al., 2012 | Larva: (1:200) S2: (1:400) | |
Antibody | anti-HRP Alexa-Fluor 647 (goat polyclonal) | Jackson ImmunoResearch Labs | Jackson ImmunoResearch Labs Cat# 123-605-021, RRID:AB_2338967 | (1:200) |
Antibody | Anti-HA (mouse monoclonal) | Biolegend | BioLegend Cat# 901533, RRID:AB_2801249 | (1:1000) |
Antibody | Anti-BetaTubulin (mouse monoclonal) | DSHB, University of Iowa, USA | DSHB Cat# E7, RRID:AB_528499 | (1:1000) |
Antibody | Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# 31430, RRID:AB_228307 | (1:2000) |
Antibody | Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# 32460, RRID:AB_1185567 | (1:2000) |
Antibody | Alexa-Fluor anti-mouse 488 (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A32723, RRID:AB_2633275 | (1:500) |
Antibody | Alexa Fluor anti-guinea pig 555 (goat polyclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# A-21435 RRID:AB_2535856 | (1:400) |
Antibody | Atto 594 conjugated anti-mouse (goat polyclonal) | Sigma-Aldrich | Sigma-Aldrich Cat# 76,085 | (1:100) |
Antibody | Abberior STAR 635P (goat polyclonal) | Abberior | Abberior Cat# ST635P-1002-500 UG, RRID:AB_2893229 | (1:100) |
Chemical compound, drug | Bouin’s fixative | Sigma-Aldrich | HT-10132 | |
Chemical compound, drug | Ethanol | Merck | CAS# 64-17-5 | |
Chemical compound, drug | ProLong Gold Antifade | Thermo Fisher Scientific | P36930 | |
Chemical compound, drug | Philanthotoxin-433 | Santa Cruz Biotechnology | Cat# sc-255421 | |
Chemical compound, drug | Schneider’s Drosophila medium | Gibco | Cat# 21720024 | |
Chemical compound, drug | FuGENE HD | Promega | Cat# E2311 | |
Chemical compound, drug | Paraformaldehyde | Merck | HT501128 | |
Chemical compound, drug | NP-40 | Thermo Fisher Scientific | 85,125 | |
Chemical compound, drug | Deoxycholate | Sigma-Aldrich | D6750 | |
Chemical compound, drug | cOmplete | Sigma-Aldrich | 11697498001 | |
Chemical compound, drug | ECL Prime Western Blotting Detection Reagent | GE Healthcare | Cat# 28980926 | |
Cell line (D. melanogaster) | Drosophila Schneider 2 (S2) Cells | Thermo Fisher Scientific | Cat# R69007 | |
Commercial assay, kit | Nitrocellulose membrane | Amersham Hibond GE Healthcare | Cat# 88,018 | |
Recombinant DNA reagent | pMT-Gal4 | Addgene | RRID:Addgene_53366 | |
Software, algorithm | Fiji / ImageJ | https://fiji.sc | RRID:SCR_002285 | Version 1.51n |
Software, algorithm | Clampex | Axon CNS, Molecular Devices | RRID:SCR_011323 | |
Software, algorithm | Leica Application Suite X | Leica Microsystems | RRID:SCR_013673 | |
Software, algorithm | Huygens Software | https://svi.nl/HuygensSoftware | RRID:SCR_014237 | |
Software, algorithm | Igor Pro | WaveMetrics | RRID:SCR_000325 | Version 6.37 |
Software, algorithm | NeuroMatic | Rothman and Silver, 2018 | RRID:SCR_004186 | Version 3.0c |
Software, algorithm | NumPy | https://www.numpy.org | RRID:SCR_008633 | |
Software, algorithm | SciPy | https://www.scipy.org | RRID:SCR_008058 | |
Software, algorithm | IPython | http://ipython.org | RRID:SCR_001658 | |
Software, algorithm | Neo | http://neuralensemble.org/neo | RRID:SCR_000634 | |
Software, algorithm | Shapely | (Gillies, 2007) https://github.com/shapely/shapely | ||
Software, algorithm | RStudio | (R Studio Team, 2020) http://www.rstudio.com/ | RRID:SCR_000432 | Version 2021.09.0 |
Software, algorithm | pwr-package | (Champely, 2020) https://github.com/heliosdrm/pwr | ||
Software, algorithm | GNU Image Manipulation Program | https://www.gimp.org/ | RRID:SCR_003182 | Version 2.8.10 |
Software, algorithm | Inkscape | http://www.inkscape.org | RRID:SCR_014479 | Version 0.92.2 |
Software, algorithm | Affinity Designer | https://affinity.serif.com/en-us/designer/ | RRID:SCR_016952 | Version 1.10.4 |
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.