Journal: Cell reports

Article Title: Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution

doi: 10.1016/j.celrep.2022.110827

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse IgG2b anti-Syt1 , Developmental Studies Hybridoma Bank , Cat#mab48-c; RRID:AB_2199314.

Techniques: Recombinant, Generated, Software

Journal: Cell reports

Article Title: Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution

doi: 10.1016/j.celrep.2022.110827

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse IgG2b anti-Syt1 , Developmental Studies Hybridoma Bank , Cat#mab48-c; RRID:AB_2199314.

Techniques: Recombinant, Generated, Software

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative anti-SYT1 and anti-CRE immunoblot of WT, Syt1 KO (generated using 1x CRE lentivirus at day 1), and neurons transduced with 10x CRE lentivirus at 13 DIV. Total DIV, CRE dose, and incubation time post CRE transduction are labeled at the top of the blot. Trichloroethanol (TCE) in-gel fluorescence served as a loading control for each immunoblot in this, and all subsequent, figures. ( b ) Cleavage was quantified via densitometry of the SYT1 signals in panel ( a ), data were fitted with a single exponential function (R 2 = 0.7583), yielding a τ for SYT1 turnover of 3.85 days. The plateau represents a significant population of long-lived SYT1 that was not turned-over. Mean +/- SEM from three trials. ( c ) Representative confocal fluorescent ICC images from mouse hippocampal neurons at 20 DIV. Images of WT, S1KO, and neurons transduced with 4x, 10x, and 20x CRE lentivirus at 13 DIV, stained with anti-MAP2 (yellow) and anti-CRE (magenta) antibodies. ( d ) Percentage of CRE positive MAP2 positive soma from indicated conditions (n = 10 for each condition from three independent trials). ( e ) Schematics of auxin-induced degron expression vectors. ( f ) Representative anti-SYT1 immunoblot from cultures transduced with vectors shown in e ii; leak prevented detectable expression of the fusion protein, even in the presence of the osTIR inhibitor, auxinole.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Western Blot, Generated, Transduction, Incubation, Labeling, Fluorescence, Staining, Expressing

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative anti-SYT1 immunoblot of endogenous and Auxin Induced Degron (AID) modified SYT1 from rat hippocampal neurons at 14 DIV. ( b ) Representative anti-FLAG immunoblot of the SYT1-mAID fusion protein and osTIR1 (both tagged with 1x FLAG) from rat hippocampal neurons at 15 DIV. The anti-FLAG blot was used to determine ~1:1 expression of osTIR1 and the SYT1-mAID fusion protein.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Western Blot, Modification, Expressing

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Illustration of the SYT1 knockoff protocol. At 14–21 DIV, inhibitor was removed from neuronal cultures and experiments were performed. Following inhibitor washout, cleavage reactions can occur in cis or trans , and the resulting cleavage product is degraded via the N-end rule. ( b ) Representative anti-SYT1, anti-synaptophysin 1 (SYP1), anti-synaptobrevin 2 (SYB2), anti-synaptogyrin 1 (GYR1) immunoblots of WT, SYT1 KO (generated using a CRE virus), and KO neurons expressing S1-SELF, in mouse hippocampal neurons at 14 DIV. (Ø) denotes a condition in which cultures have never been exposed to PRV. ( c ) Self-cleavage time course of S1-SELF, upon PRV washout. Cleavage was quantified via densitometry of the SYT1 immunoblots in panel b , and plotted. Mean +/- SEM from three independent trials are shown, and the time constant was determined by fitting the data with a single exponential function (R 2 = 0.9498). The τ for S1-SELF cleavage was 2 hr 45 min. ( d ) Schematic of S1-SELF; domains are labeled.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Western Blot, Generated, Expressing, Labeling

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative anti-SYT1 immunoblot of mouse hippocampal neurons at 14 DIV showing: 1) CRE excision of Syt1 , 2) stable expression of SYT1-SELF + 0.5 µM PRV, and 3) complete cleavage and degradation of SYT1-SELF in the absence of PRV. (Ø) denotes a condition in which cultures have never been exposed to PRV. These experiments follow the protocol that is detailed in . ( b ) Immunoblot of msGFP comparing expression levels of two different NS3/4A containing constructs using original codons and optimized codons (construct 1: mito-NS3/4A, construct 2: ER-NS3/4A). Constructs were identically prepped and transduced, using lentivirus, into rat cortical neurons. ( c ) Relative adaptiveness (wij) of codons from msGFP, original NS3/4A, and NS3/4A codon optimized transcript. msGFP CAI-value = 0.630283. Note how this high value contrasts with the original NS3/4A transcript. NS3/4A original CAI-value = 0.276799 and NS3/4A codon optimized CAI-value = 0.621645. ( d ) Diagram of the synaptophysin self-cleaving construct, denoted SYP1-SELF. The protease cleavage site was inserted between the fourth TMD and the carboxy termini YG(P/Q) repeats, denoted in grey and with an X. ( e ) Representative anti-Flag immunoblot showing time-course of cleavage for SYP1-SELF in 0.5 µM PRV.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Western Blot, Expressing, Construct

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative super-resolution fluorescent ICC images from mouse hippocampal neurons at 15 DIV. Images of WT, S1KO, and neurons expressing S1-SELF, stained with anti-synaptophysin (cyan), anti-SYT1 luminal domain (LD) (yellow), and anti-SYT1 C2A domain (C2A) (magenta) antibodies; in the last column, all three signals were merged. Note the SYT1 LD antibody recognizes the rat luminal domain with much higher affinity than the mouse; therefore, in the WT example, only a trace signal is detected. All images were acquired using the same microscope settings, and all samples were prepared in parallel. Scale bar, 5 µm. ( b ) Illustration of S1-SELF, and the antibodies used for ICC. ( c ) Pearson’s correlation coefficient (PCC) plot, measured using JaCoP for ImageJ . As S1-SELF is cleaved, the PCC of the synaptophysin to SYT1 C2A signal decreases (Mean PCC +/- SEM are plotted, n = 10 for each condition from three independent trials).

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Expressing, Staining, Microscopy

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Representative, individual ROI (black) and average (green) iGluSnFR traces from a single-field stimulus, recorded optically at 100 Hz. The examples shown are from a single FOV for each condition. ( b ) Histograms of iGluSnFR (ΔF/F 0 ) peaks plotted using 10 ms bins. Peaks were binned over the entire 1.5 s of recording; a 0.5 s epoch, just before and after the stimulus, is shown. Samples were color-coded as follows: WT (black), Syt1 KO (+CRE) (red), S1KO + S1-SELF + 0.5 µM PRV (green); the S1-SELF PRV washout samples were: 2 (purple), 4 (blue), 6 (orange), and 8 hr (grey). The histograms include all combined data from four independent trials, with 10 to 16 FOVs for each group. Comparisons between all conditions, and statistical analysis, are provided in . ( c ) Representative images showing temporally color-coded max projections (time projection) of iGluSnFR ΔF/F 0 peaks 100 ms after a single stimulus. Temporal color code is from red (time of stimulation) to purple (100 ms after the stimulus). Scale bar, 5 µm. Figure 5—source data 1. Loss of SYT1 via knockoff disrupts synchronous release. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for the histograms shown in .

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques:

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Average, normalized, traces of iGluSnFR ΔF/F 0 from . Samples are color coded as follows: WT (black), Syt1 KO (+CRE) (red), S1KO + S1-SELF + 0.5 µM PRV (green), S1-SELF 2 hr washout (purple), S1-SELF 4 hr washout (blue), S1-SELF 6 hr washout (orange), and S1-SELF 8 hr washout (grey). Values from four independent trials, with 10 to 16 field of views for each group.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques:

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) mEPSC rates in WT (black), S1KO (red), and neurons expressing S1-SELF (+ PRV green and −4 hr PRV blue). Values are mean +/- SEM, 12 to 18 neurons per condition (n). Comparisons between conditions and statistical analysis, are provided in . ( b ) Representative traces from ( a ). ( c ) Average mEPSC amplitude. ( d ) Average mEPSC 10–90% half-width. No statistical differences between groups in ( c ) or ( d ) were observed. ( e ) Representative images showing temporally color-coded max projections (time projection) of iGluSnFR ΔF peaks. mGT denotes miniature glutamate release events. Temporal color code is from red (start of image acquisition) to purple (60 s after image acquisition start). Scale bar, 5 µm. ( f ) mGT rates in WT, S1KO, and neurons expressing S1-SELF, recorded optically with iGluSnFR. Values are mean +/- SEM from three independent trials, with 13 to 21 field of views for each group. Comparisons between conditions and statistical analysis, are provided in . ( g ) Percentage of iGluSnFR ΔF/F 0 peaks within 10 ms following a single stimulus (data are from ). Values are mean +/- SEM from four independent trials, with 10 to 16 field of views for each group. Comparisons between conditions and statistical analysis, are provided in . ( h ) Inverse correlation between synchronous release (≤10 ms) and spontaneous (mGT) release in WT, S1KO, and S1-SELF neurons. Data were fitted using a linear regression; r 2 = 0.7671, p-value=0.0097. Figure 6—source data 1. Acute knockoff of SYT1 unclamps spontaneous release as determined electrophysiologically. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for data in . Figure 6—source data 2. Acute knockoff of SYT1 unclamps spontaneous release via iGluSnFR. Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for data in . Figure 6—source data 3. Acute knockoff of SYT1 decreases synchronous release (increases asynchronous release). Table summarizing the Kruskal-Wallis test and Dunn’s multiple comparison test for histogram data in .

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Expressing

Journal: eLife

Article Title: Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

doi: 10.7554/eLife.56469

Figure Lengend Snippet: ( a ) Example trace of background-subtracted, iGluSnFR ΔF spontaneous events, from cultured hippocampal neurons expressing S1-SELF, 6 hr after washout of PRV; four spontaneous events were detected (green arrows). ( b ) Histogram of background ROI peak ΔF (grey) compared to manually identified, ROI ΔF peaks (green) of spontaneous events. Background peaks are centered at 3 a.u., while mGT events are centered at 9 a.u., n = 141 for each group. ( c ) The peak amplitude of mGT events (green, panel C) and random ROIs (grey), plotted against the peak signal-to-noise ratio at each ROI. This graph indicates that low-intensity events were identified because the background noise, for those specific events, was also low, n = 141 for each group. ( d ) Representative anti-CDK5 immunoblot of WT, Syt1 KO (generated using a CRE virus), and KO neurons expressing S1-SELF. (Ø) PRV means that the culture has never seen inhibitor (PRV). (Ø) denotes a condition in which cultures have never been exposed to PRV.

Article Snippet: Primary antibodies were: anti-CRE (1:100, 2D8) (Millipore; MAB3120, RRID: AB_2085748 ), anti-MAP2 (1:100) (Millipore; AB15452, RRID: AB_805385 ), anti-MAP2 (1:500) (Millipore; AB5543, RRID: AB_571049 ), anti-PSD95 (1:250) (ThermoFisher; MA1-046, RRID: AB_2092361 ), anti-vGlut1 (1:500) (Millipore; AB5905, RRID: AB_2301751 ), anti-SYT1 C2A (1:50, 48) (DSHB; mAB 48; RRID: AB_2199314 ), anti-SYT1 luminal domain (LD) (1:200) (SySy; 105 103; RRID: AB_11042457 ), and anti-synaptophysin (SYP) (1:500) (SySy; 101 004; RRID: AB_1210382 ).

Techniques: Cell Culture, Expressing, Western Blot, Generated

Journal: Current biology : CB

Article Title: Live Observation of Two Parallel Membrane Degradation Pathways at Axon Terminals

doi: 10.1016/j.cub.2018.02.032

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-Syt1 , Developmental Studies Hybridoma Bank (DSHB) , 3H2 2D7, RRID: AB_528483.

Techniques: Transduction, Recombinant, Plasmid Preparation, Software