1. Cell Biology
  2. Neuroscience

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

  1. Shataakshi Dube O'Neil
  2. Bence Rácz
  3. Walter Evan Brown
  4. Yudong Gao
  5. Erik J Soderblom
  6. Ryohei Yasuda
  7. Scott H Soderling  Is a corresponding author
  1. Department of Neurobiology, Duke University Medical Center, United States
  2. Department of Anatomy and Histology, University of Veterinary Medicine, Hungary
  3. Department of Cell Biology, Duke University Medical Center, United States
  4. Proteomics and Metabolomics Shared Resource and Center for Genomic and Computational Biology, Duke University Medical Center, United States
  5. Max Planck Florida Institute for Neuroscience, United States
https://doi.org/10.7554/eLife.63756
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Cite this article
  1. Shataakshi Dube O'Neil
  2. Bence Rácz
  3. Walter Evan Brown
  4. Yudong Gao
  5. Erik J Soderblom
  6. Ryohei Yasuda
  7. Scott H Soderling
(2021)
Action potential-coupled Rho GTPase signaling drives presynaptic plasticity
eLife 10:e63756.
https://doi.org/10.7554/eLife.63756
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9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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Identification of the proteomic composition of the presynaptic cytomatrix using in vivo BioID.

(A) Schematic of the iBioID approach in presynaptic terminals. (B) Timeline of in vivo injections and sample collection. (C) Filters used to select proteins based on fold enrichment over negative …

Figure 1—source data 1

The Synapsin iBioID proteome.

https://cdn.elifesciences.org/articles/63756/elife-63756-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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Validation of Synapsin iBioID probes in cultured hippocampal neurons.

(A–C) Representative images of localization (HA or GFP; green), biotinylation (Streptavidin; magenta), and a presynaptic marker (Bassoon; blue) in neurons expressing (A) BioID2-Linker-Synapsin1a-HA, …

Figure 2
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Validation of the presynaptic localization of Synapsin iBioID proteins.

(A) Schematic of approach to tag endogenous proteins in neurons using HiUGE. Cultured hippocampal neurons were infected on DIV0 with AAVs containing the candidate sgRNA and a 2x-HA-V5-Myc HiUGE …

Figure 2—source data 1

Candidate genes screened for HiUGE validation of the Synapsin iBioID proteome.

https://cdn.elifesciences.org/articles/63756/elife-63756-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
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Actin signaling pathways in presynaptic terminals.

(A) Network showing the diversity of presynaptic actin signaling pathways in the Synapsin iBioID proteome. Node titles correspond to gene name, and node size emphasizes the proteins further studied. …

Figure 3—figure supplement 1
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Additional analysis for immunogold electron microscopy.

(A) Four large overview images of Rac1 immunogold labeling in mouse hippocampal CA1. Scale bars, 1000 nm. (B) Distribution of presynaptic Rac1 gold particles among synaptic vesicles, plasma …

Figure 4 with 3 supplements
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Presynaptic Rac1 negatively regulates synaptic vesicle replenishment.

(A) Schematic of mixed hippocampal neuron cultures to isolate effects of presynaptic Rac1 knockout. Whole-cell patch clamp recordings were conducted on tdTomato+ WT neurons with light delivered …

Figure 4—figure supplement 1
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Characterization of neuronal cultures used for electrophysiology.

(A) Representative image of an entire coverslip used for presynaptic isolation. Presynaptic neurons expressing an opsin (ChR2-EYFP; green) were sparsely seeded amongst other neurons (NeuN; magenta). …

Figure 4—figure supplement 2
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Single evoked currents and asynchronous release in Rac1 neurons.

(A) Traces of all analyzed EPSCs in presynaptic Rac1 KO experiments. (B) Examples of rejected responses contaminated by ‘polysynaptic’ or recurrent events. (C–D) Visual representation of …

Figure 4—figure supplement 3
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Comparison of optogenetic and electrical stimulation in elevated extracellular calcium.

(A) Schematic of mixed hippocampal cultures. Whole-cell patch clamp recordings were conducted on non-fluorescent WT neurons with light delivered through the objective by a 460 nm LED and electrical …

Figure 5 with 3 supplements
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Presynaptic Arp2/3 negatively regulates release probability and synaptic vesicle replenishment.

(A) Schematic of mixed hippocampal neuron cultures to isolate effects of presynaptic Arpc3 knockout. (B) Representative images of WT and KO cultures fixed on DIV16 and stained for ChR2-EYFP (blue), …

Figure 5—figure supplement 1
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Single evoked currents and asynchronous release in Arpc3 neurons.

(A) Quantification of EPSCs in Arpc3 cultures (WT n=10/3, KO n=10/3) for amplitude (t18=2.323, p=0.0321), charge (t18=2.127, p=0.0475), rise time (U=38, p=0.3811), and decay time constant (U=17, …

Figure 5—figure supplement 2
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ArpC3 loss does not affect the density of synapses formed along axons.

(A) Schematic of mixed hippocampal neuron cultures to isolate effects of Arpc3 knockout on axonal synapse density. Arpc3fl/fl;Ai14 neurons were electroporated with tdTomato and sparsely seeded …

Figure 5—figure supplement 3
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Action potential firing and intrinsic membrane properties in Rac1 and Arpc3 neurons.

(A) Schematic of Rac1 mixed hippocampal neuron cultures. Current clamp recordings were conducted from ChR2+ neurons with light delivered through the objective by a 460 nm LED (WT n=12 neurons/3 …

Figure 6 with 1 supplement
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Rac1 alters vesicle replenishment specifically at presynaptic terminals.

(A) Schematic of hippocampal cultures expressing presynaptic Rac1 inhibitory peptide (W56) or scrambled control (Scr). Whole-cell patch clamp recordings were conducted with local electrical …

Figure 6—figure supplement 1
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Single evoked currents and asynchronous release with presynaptic Rac1 inhibition.

(A) Quantification of EPSCs in cultures with presynaptic Rac1 inhibitory peptide (Scr n=17/3, W56 n=17/3) for amplitude (t32=0.3037, p=0.7634), charge (U=126, p=0.5401), rise time (U=125, p=0.5177), …

Figure 7
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Arp2/3 loss occludes replenishment rate changes by presynaptic Rac1.

(A) Schematic of mixed hippocampal cultures to inhibit presynaptic Rac1 in Arpc3 knockout neurons. Whole-cell patch clamp recordings were conducted on non-fluorescent WT neurons with light delivered …

Figure 8
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Bidirectional control of presynaptic Rac1 signaling modulates short-term synaptic depression.

(A) Schematic of constructs created to control the firing of presynaptic neurons with reduced or enhanced Rac1 signaling. ChrimsonR-tdTomato was expressed alone as a control (WT), or co-expressed …

Figure 9 with 1 supplement
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Action potential trains activate Rac1 in presynaptic terminals.

(A) Experimental design in organotypic hippocampal slices. (B) Schematic of Rac1 sensor. Activation of Rac1 leads to its association with the GTPase-binding domain of Pak2R71C,S78A (PBD2), …

Figure 9—figure supplement 1
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Presynaptic boutons in organotypic slices used for 2pFLIM contain Synapsin.

(A) Representative image of an organotypic hippocampal slice microinjected in CA3 with AAV-mEGFP-Rac1 and AAV-mCherry-PBD2-mCherry on DIV10 and fixed and stained on DIV20 for DAPI (blue), GFP …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C57BL/6JThe Jackson LaboratoryCat# 000664; RRID:IMSR_JAX:000664Both sexes used
Genetic reagent (Mus musculus)H11Cas9The Jackson LaboratoryCat# 028239; RRID:IMSR_JAX:028239Both sexes used
Genetic reagent (Mus musculus)Rac1fl/flChrostek et al., 2006Both sexes used
Genetic reagent (Mus musculus)Arpc3fl/fl;Ai14 (Rosa26)Kim et al., 2015Both sexes used
Biological sample (Mus musculus)Primary hippocampal neuron culturesThis paperFreshly isolated from P0-P1 Mus musculus brains
Biological sample (Mus musculus)Organotypic hippocampal slicesThis paperFreshly isolated from P3-P8 Mus musculus brains
Cell line
(Homo sapiens)		HEK293TATCCCat# CRL-3216; RRID:CVCL_0063
AntibodyAnti-HA (clone 3F10) (rat monoclonal)RocheCat# 11867431001; RRID:AB_390919ICC (1:500)
AntibodyAnti-HA (clone 16B12) (mouse monoclonal)BiolegendCat# 901501; RRID:AB_2565006ICC (1:500)
AntibodyAnti-V5 (mouse monoclonal)ThermoFisherCat# R960-25; RRID:AB_2556564ICC (1:500)
AntibodyAnti-Myc (clone 9E10) (mouse monoclonal)Santa CruzCat# sc-40; RRID:AB_627268ICC (1:250)
AntibodyAnti-Bassoon (clone SAP7F407) (mouse monoclonal)AbcamCat# ab82958; RRID:AB_1860018ICC (1:400)
AntibodyAnti-GFP (chicken polyclonal)AbcamCat# ab13970; RRID:AB_300798ICC, IHC (1:500)
AntibodyAnti-RFP (rabbit polyclonal)RocklandCat# 600-401-379; RRID:AB_2209751ICC, IHC (1:500)
AntibodyAnti-RFP (clone 5F8) (rat monoclonal)ChromotekCat# 5f8-100;
RRID:AB_2336064		ICC (1:500)
AntibodyAnti-Homer1 (rabbit polyclonal)Synaptic SystemsCat# 160 002;
RRID:AB_2120990		ICC (1:500)
AntibodyAnti-Synapsin1 (guinea pig polyclonal)Synaptic SystemsCat# 106 104;
RRID:AB_2721082		ICC, IHC (1:500)
AntibodyAnti-Vgat (guinea pig polyclonal)Synaptic SystemsCat# 131 004;
RRID:AB_887873		ICC (1:500)
AntibodyAnti-Gephyrin (mouse monoclonal)Synaptic SystemsCat# 147 011;
RRID:AB_887717		ICC (1:300)
AntibodyAnti-NeuN (clone 1B7) (mouse monoclonal)AbcamCat# ab104224; RRID:AB_10711040ICC (1:1000)
AntibodyAnti-Rac1 (clone 102) (mouse monoclonal)BD BiosciencesCat# 610650;
RRID:AB_397977		EM (1:100),
ICC (1:250)
AntibodyAnti-ArpC2 (rabbit polyclonal)MilliporeSigmaCat# 07–227;
RRID:AB_310447		EM (1:200)
AntibodyStreptavidin Alexa Fluor 555ThermoFisherCat# S-32355; RRID:AB_2571525ICC (1:500)
AntibodyNanogold-StreptavidinNanoprobesCat# 2016EM (1:100)
Recombinant DNA reagentpCMV-EGFP-Synapsin1a (rat)Gitler et al., 2004bfrom George Augustine
Recombinant DNA reagentpAAV-hSyn-hChR2(H134R)-EYFPAddgeneCat# 26973; RRID:Addgene_26973
Recombinant DNA reagentpCAG-ChrimsonR-tdTomatoKlapoetke et al., 2014; AddgeneCat# 59169; RRID:Addgene_59169
Recombinant DNA reagentpAAV-hSyn-CaMPARI2Moeyaert et al., 2018; AddgeneCat# 101060; RRID:Addgene_101060
Recombinant DNA reagentpAAV-hSyn-BioID2-HAThis papersee Plasmids
Recombinant DNA reagentpAAV-hSyn-BioID2-Linker-Synapsin1a-HAThis papersee Plasmids
Recombinant DNA reagentpAAV-HiUGE-GS-gRNA vectorsThis papersee Figure 2—source data 1
Recombinant DNA reagentpAAV-HiUGE-2xHA-V5-Myc donor vectorsThis papersee HiUGE tagging
of candidate genes
Recombinant DNA reagentpAAV-hSyn-CreThis papersee Plasmids
Recombinant DNA reagentpAAV-hSyn-W56-GFP-Linker-Synapsin1aThis papersee Plasmids
Recombinant DNA reagentpAAV-hSyn-Scr-GFP-Linker-Synapsin1aThis papersee Plasmids
Recombinant DNA reagentpCAG-ChrimsonR-tdTomato-P2A-HA-PA Rac1 (DN)This papersee Plasmids
Recombinant DNA reagentpCAG-ChrimsonR-tdTomato-P2A-HA-PA Rac1 (CA)This papersee Plasmids
Recombinant DNA reagentpAAV-hSyn-mEGFP-Rac1This papersee Plasmids
Recombinant DNA reagentpAAV-hSyn-mCherry-PBD2-mCherryThis papersee Plasmids
Commercial assay or kitMouse neuron nucleofector kitLonzaCat# VPG-1001
Commercial assay or kitPierce Protein A agarose resinThermoFisherCat# 20333
Commercial assay or kitPierce high capacity NeutrAvidin agarose resinThermoFisherCat# 29202
Commercial assay or kitS-Trap micro kitProtifiCat# K02-micro-10
Commercial assay or kitIntensEMGE Healthcare Life SciencesCat# RPN491
Commercial assay or kitGoldEnhance EM PlusNanoprobesCat# 2114
Chemical compound, drugBiotinMilliporeSigmaCat# B4501;
CAS: 58-85-5
Chemical compound, drugSrCl2MilliporeSigmaCat# 255521;
CAS: 10025-70-4
Chemical compound, drugTetrodotoxin citrate (TTX)AbcamCat# ab120055;
CAS: 18660-81-6
Chemical compound, drugCdCl2MilliporeSigmaCat# 202908;
CAS: 10108-64-2
Software, algorithmImageJ (FIJI)Schindelin et al., 2012; Schneider et al., 2012RRID:SCR_002285Version 1.29, Version 1.52a
Software, algorithmPuncta Analyzer plugin for ImageJ by Barry WarkIppolito and Eroglu, 2010
Software, algorithmSimple Neurite Tracer plugin for ImageJLongair et al., 2011RRID:SCR_016566
Software, algorithmProteome DiscovererThermo ScientificRRID:SCR_014477Version 2.2
Software, algorithmMascot Distiller and Mascot ServerMatrix ScienceRRID:SCR_000307Version 2.5
Software, algorithmCytoscapeCytoscape ConsortiumRRID:SCR_003032Version 3.6
Software, algorithmgetPPIs R packageBradshaw, 2021http://github.com/twesleyb/getPPIs
Software, algorithmDAVID bioinformatics toolsDennis et al., 2003RRID:SCR_001881https://david.ncifcrf.gov
Software, algorithmEnsembl genome browserZerbino et al., 2018RRID:SCR_002344http://uswest.ensembl.org
Software, algorithmCRISPOR guide RNA selection toolHaeussler et al., 2016RRID:SCR_015935http://crispor.tefor.net
Software, algorithmpClampMolecular DevicesRRID:SCR_011323Version 10
Software, algorithmMiniAnalysisSynaptosoftRRID:SCR_002184
Software, algorithmMATLABMathWorksRRID:SCR_001622Version R2017a
Software, algorithmPrismGraphPadRRID:SCR_002798Version 8.4

Additional files

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