halotag into porange  (Addgene inc)

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative super-resolution fluorescent immunocytochemistry (ICC) image of rat hippocampal neurons at 15 days in vitro (DIV) expressing uniformly transduced LAMP1-msGFP and sparsely transduced, untagged SYT7α. These neurons were fixed and stained with antibodies to SYT7 (juxta-membrane region) and the axon initial segment (AIS) (anti pan-neurofascin). Scale bar = 5 μm. ( b ) Representative super-resolution images of cytosolically expressed mRuby3 (yellow/top left), SYT7α-HaloTag/JF646 (magenta/top right), and merged (bottom left). Scale bar = 5 μm. ( c ) Quantification of the ratio between fluorescent channels. Axonal ratio of SYT7-HaloTag:mRuby3 signal is 0.61 +/- 0.06, n = 30, while dendritic ratio is 0.21 +/- 0.01. Values are means +/- SEM from two independent experiments; p-value <0.0001 using unpaired two-tailed Welch’s t-test. ( d ) Representative super-resolution optical slice of an axon (identified via morphology) expressing cytosolic mRuby3 (yellow) and SYT7α-HaloTag/JF646 (magenta). Merged image also denotes the line used in panel ( e ). Scale bar = 1 μm. ( e ) Plot of the normalized intensity profile along the orange dashed line in panel ( d ). ( f ) Representative super-resolution optical slice of a somatic lysosome from a rat hippocampal neuron at 16 DIV expressing LAMP1-msGFP (cyan), SYT7α-HaloTag/JF549 (yellow), and incubated with 0.5 μM Prosense 680 (magenta) for 12 hr. Scale bar = 1 μm. Merged image also denotes the line used in panel ( g ). ( g ) Plot of the normalized intensity profile along the dashed orange line in panel ( f ).

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Immunocytochemistry, In Vitro, Expressing, Staining, Two Tailed Test, Incubation

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative z-stack image of rat hippocampal neurons at 15 DIV transfected with a PM-msGFP P2A SYT7α-HaloTag construct, fixed and stained with HTL-JF646. As with Figure (3a), asymmetric axonal enrichment (indicated by white arrows), versus dendrites, is readily apparent. Scale bar = 10 μm. ( b ) Graph of the normalized intensity profile along the vertical white line drawn over dendrites and the axon in panel ( a ). Note: this is transient overexpression, so SYT7α-HaloTag is observable on the dendritic plasma membrane, but is still comparatively enriched in the axon. ( c ) Additional super-resolution optical slice of an axon (axonal varicosities) expressing cytosolic mRuby3 (yellow) and SYT7α-HaloTag (magenta). Merged image also denotes the line used in panel ( d ). ( d ) Graph of the normalized intensity profile along the line. Scale bar = 1 μm. ( e ) Representative max z-stack of a neuron from a coverslip exposed to SYT7 homology-independent targeted integration (HITI) lentiviruses. Neurons were incubated with HTL-JF549 (yellow) and anti-AIS (cyan), fixed, and imaged using highly inclined and laminated optical sheet (HILO) microscopy because the sample signal was very dim. Signal is present primarily in axons and irregular globular structures in the soma (left inset). ( f ) Representative tiled z-stacks showing neurons from coverslips exposed to SYT7 HITI lentiviruses and imaged using Airyscan confocal microscopy after conducting anti-HaloTag immunocytochemistry (ICC) to amplify the signal. Anti-HaloTag is shown in cyan and anti-AIS is shown in magenta; white arrows point to major features. ( g ) The constructs that were used are shown in a schematic form. Constructs were based on the pORANGE vector. pORANGE vectors were designed and tested but the HITI site available for carboxy-terminal tagging of SYT7 with HaloTag has a low frequency of recombination; so this construct was split into two different pFUGW-based lentivirus constructs, made into lentivirus, and applied to neuronal cultures to achieve total coverage.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Transfection, Construct, Staining, Over Expression, Expressing, Incubation, Microscopy, Confocal Microscopy, Immunocytochemistry, Plasmid Preparation

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative anti-SYT7, anti-SYT1, and anti-synaptophysin (SYP) immunoblots of developing wild-type (WT) rat cortical neurons at the indicated DIV. Trichloroethanol (TCE) staining was used to visualize the total protein and serves as a loading control. ( b ) Quantification of normalized protein levels (αSYT7, αSYT1, αSYP, and TCE stain) from panel ( a ). Values are mean +/- SEM from four independent trials. Significant p-values between SYT7 and SYT1/SYP are labeled ****p<0.0001, **p = 0.001

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Western Blot, Staining, Labeling, Expressing, Transfection, Transduction

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative anti-SYT7 immunoblot from wild-type (WT) and SYT7KO-dissociated mouse hippocampal neurons transduced with various constructs. WT (endogenous synaptotagmin (SYT)7 is expressed at very low levels in neurons obtained from P0 mice) and SYT7KO conditions are shown as controls. SYT7α represents an overexpressed untagged SYT7 alpha isoform, while HT- SYT7α and SYT7α-HT represent amino- and carboxy-terminal-tagged SYT7 with the HaloTag enzyme. ( b ) The same blot as in ( a ) but stripped and probed with anti-HaloTag. ( c ) Representative anti-SYT7 immunoblot of rat cortical neurons expressing transduced HT-SYT7α with various combinations of a competitive γ-secretase inhibitor (DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)) and metalloprotease inhibitors GI 254023X for ADAM10, TAPI-1 for ADAM17, and Verubecestat for BACE1, with TCE staining as a load control. ( d ) The same setup as in panel ( c ) but with neurons transduced with untagged SYT7α, with TCE staining used as a load control. ( e ) Representative super-resolution optical slice of an untransduced rat hippocampal neuron. Before fixing neurons, they were incubated with 1 nM JF549i for 2 days to reveal any nonspecific labeling from the JF549i dye. Fixed neurons were decorated with anti-pan-neurofascin antibodies to mark axon initial segments.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Western Blot, Transduction, Construct, Expressing, Staining, Incubation, Labeling

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative anti-SYT7 immunoblot from rat hippocampal neurons with trichloroethanol (TCE) staining as a loading control. Conditions from left to right are blank/no protein, control conditions, neurons ttreated with 1 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (presenilin competitive inhibitor), DAPT and 20 μM GI 254023X (ADAM10 selective inhibitor), or treated with GI 254023X only, all from DIV 5 onward. ( b ) Representative anti-SYT7 immunoblot using mouse hippocampal neurons for wild-type (WT) and SYT7KO antibody controls along with rat cortical neurons grown in various concentrations of DAPT to assay half maximal inhibitory concentration (IC 50 ), with TCE staining as a loading control. ( c ) Graph of the fraction of processed synaptotagmin 7 (SYT7) when grown in various DAPT concentrations in relation to control conditions (IC 50 curve) results in an IC 50 of 71 nM. The lowest specific SYT7 band was used for quantitating cleavage and IC 50 of DAPT. Values are means +/- SD after log transformation from three independent experiments. ( d ) Cartoon illustrating the logic and methodological approach to determine whether full-length SYT7 protein transits through the plasma membrane (PM) prior to amino-terminal cleavage by γ-secretase. JF549i is a membrane-impermeant version of JF549 (JF549 and JF549i are nonfluorogenic). In (1), cleavage can take place in the post-Golgi vesicle, prior to axonal PM localization or cleavage happens at the PM. No fluorescent HaloTag is observable in this scenario. In (2), SYT7 transits through the PM before being cleaved in a synaptic endosome. Only in this scenario will fluorescent HaloTag be observable in neurons. ( e ) Representative super-resolution optical slice of a rat hippocampal neuron transduced with LAMP1-msGFP (cyan) and HaloTag-SYT7α (yellow). Before fixing neurons, they were incubated with 1 nM HTL-JF549i for 2 days. Fixed neurons were decorated with anti-pan-neurofascin (magenta) antibodies to mark the axon initial segment (AIS). White box indicates the area that is enlarged to show the detail below the image. The labels (i), (ii), and (iii) indicate areas where HTL-JF549i appears inside lysosomes, clustered on the edge of lysosomes, or completely independent of lysosomes, respectively. ( f ) Line profile from the dashed line in panel ( e ) with normalized intensity of LAMP1-msGFP (cyan) and JF549i (yellow). The labels (i) and (ii) are labeled on the line profile as well and correspond to the same labels as in panel ( e ). Cartoon schematic of the analyzed signal is above the graph.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Western Blot, Staining, Concentration Assay, Transformation Assay, Transduction, Incubation, Labeling

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Representative super-resolution maximum z-projections of rat hippocampal neurons transduced with LAMP1-msGFP (cyan), fixed for immunocytochemistry (ICC), and stained for synaptotagmin 1 (SYT1) (yellow) and the axon initial segment (AIS) (magenta). Four separate conditions were imaged: control neurons, neurons grown for 10–12 days in 0.5 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), neurons exposed to 2-bromopalmitate (2-BP) for 3 hr before imaging, and neurons exposed to a combination treatment of DAPT and 2-BP. ( b ) Same as in panel ( a ), but instead of anti-SYT1 staining, neurons were transduced with SYT7α-HaloTag and reacted with JF549 during overnight primary antibody incubation to monitor SYT7α localization. Scale bar = 10 μm. ( c ) Illustration of the model neuron and compartments assayed for SYT7α-HaloTag colocalization. ( d ) Bar graph showing changes in colocalization of SYT7α-HaloTag/JF549 and labeled organelles (M6PR and sortilin label post-Golgi vesicles). Quantified by taking the difference of the PCC between DAPT-treated and control neurons in each condition. Values are means +/- error propagated SEM from at least three separate experiments for each condition. ( e ) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and when grown in 0.5 μM DAPT. Cultures were labeled with JF635 at 13 DIV and then robustly washed with conditioned media. The disappearance of labeled SYT7α-HaloTag/JF635 from the gel can be used to calculate protein half-life. Control SYT7α-HaloTag/JF635 runs between 75 and 100 kDa, while DAPT-treated SYT7α-HaloTag/JF635 runs slightly higher because cleavage of the amino-terminus is blocked. Trichloroethanol (TCE) staining was used as a loading control. ( f ) Normalized intensity of SYT7α-HaloTag/JF635 plotted as the fraction of total control SYT7α-HaloTag/JF635 against days post-wash. Values are means +/- SEM from three independent experiments. Single exponential functions were fitted to control (black) and DAPT (red) conditions. The tau for control SYT7α-HaloTag/JF635 is 9.5 days, while the tau for DAPT-treated SYT7α-HaloTag/JF635 is 3.3 days.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Transduction, Immunocytochemistry, Staining, Imaging, Incubation, Labeling, Fluorescence

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Average iGluSnFR ΔF/F 0 traces from high-frequency stimulation (HFS) of wild-type (WT) (black, n = 9) and WT + 0.5 μM DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (gray, n = 11)-treated mouse hippocampal neurons, from two independent experiments. Samples were field stimulated at 20 Hz for 2.5 s (50 action potentials (APs)). ( b ) Fraction of active synapses (synapses releasing peak glutamate above baseline, >4 SD above noise) as a function of stimulation number during HFS. Values are means +/- SEM, p = ns by two-way analysis of variance (ANOVA) comparing genotypes. ( c ) Synchronous fraction of iGluSnFR ΔF/F 0 peaks (synchronous peaks within 10 ms of stimulus) as a function of stimulation number during HFS. Values are means +/- SEM, ****p<0.0001 by two-way ANOVA comparing genotypes. ( d ) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and when grown in 0.5 μM DAPT. ( e ) Representative in-gel fluorescence of the protein extracted from rat cortical neurons transduced with palmitoylation site mutant (3x Cys -> 3x Ala) SYT7α-HaloTag and pulse-chased with JF635 at 13 DIV under control conditions and then grown in 0.5 μM DAPT. Palmitoylation mutants are not stable in control conditions, presumably because they are cleaved by γ-secretase and degraded.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Fluorescence, Transduction, Mutagenesis

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a–c ) Representative super-resolution maximum z-projection of rat hippocampal neurons transduced with ( a ) a plasma membrane-targeted synaptotagmin (SYT)7α, [PM-SYT7α-HaloTag (magenta)], plus LAMP1-msGFP (cyan), ( b ) a lysosome-targeted SYT7α, [LAMP1-SYT7α-HaloTag (magenta)], plus LAMP1-msGFP (cyan), and ( c ) a synaptic vesicle-targeted SYT7α, [SYP-SYT7α-HaloTag (magenta)]. Neurons were fixed and stained with HTL-JF635 ( a–c ), anti-pan-neurofascin (yellow, a, b ), and anti-SYT1 (yellow, c ) antibodies. For panel ( c ), a blank image is included to preserve the layout. For panels ( a ) and ( b ), SYT7α constructs were sparsely transduced to better examine localization. Scale bars = 10 μm. ( d ) Depression plot, showing the fraction of active synapses (synapses releasing peak glutamate above baseline, >4 SD above noise) as a function of stimulation number during high-frequency stimulation (HFS). Values are means (solid line) +/- SEM (shaded error), WT (black, n = 15), SYT7KO (red, n = 13), and SYT7α rescue (green, n = 15) from three independent experiments; SYT7KO vs SYT7α rescue is ****p<0.0001 by two-way analysis of variance (ANOVA) comparing genotypes. ( e ) Depression plot from panel ( d ) but with SYT7α rescue constructs included. Values are means (solid line) +/- SEM (shaded error), PM-SYT7α rescue (blue, n = 15), LAMP1-SYT7α rescue (orange, n = 15), and SYP-SYT7α rescue (purple, n = 15) from three independent experiments. ( f ) Multiple comparison confidence interval (95% CI) plot from data in panel ( e ). Plot was generated from two-way ANOVA comparing the predicted mean difference between genotypes of normalized active synapses. Comparisons with errors including zero are not statistically different. Total summary statistics are included in . ( g ) An X-Y plot of paired-pulse ratio (PPR) generated at 20 Hz (from first two pulses of HFS). Values are means +/- SEM, where X values are the ratio of the change in glutamate release (ΔF/F 0 iGluSnFR peaks) and Y values are the fraction of regions of interest (ROIs) releasing glutamate (active sites) from wild-type (WT) (black), SYT7KO (red), and SYT7α rescue (green), PM-SYT7α rescue (blue), LAMP1-SYT7α rescue (orange), and SYP-SYT7α rescue (purple). ( h ) Train asynchronous release (peak release recorded between 10 ms and 50 ms post-stimulus) of WT and SYT7KO vs the labeled rescue constructs. Values are means +/- SEM and are the average asynchronous values from each stimulus during a 50 action potential (AP) (20 Hz) HFS; so n = 50 for each group. All comparisons and summary statistics are provided in , and only some are labeled on the graph for presentations sake; p-values are as follows: ***p = 0.001, *p = 0.0147, by one-way ANOVA with Holm-Sidak’s multiple comparisons test. ( i ) Summary X-Y plot illustrating different magnitudes of rescue for three of the proposed functions of SYT7. Values are means +/- SEM, where X values represent depression percentage (release from 10 th to 50 th stimulation normalized to first) and Y values are the average asynchronous percentage of each genotype during the HFS train. The size of each dot reflects the relative magnitude of each PPR, normalized on a scale from the largest, 10 au (most paired-pulse facilitation (PPF)), to the smallest, 1 au (least PPF). Figure 7—source data 1. Total summary statistics from multiple comparison confidence interval (95% CI) plot from data in panel (e). Figure 7—source data 2. Statistic summary using one-way ANOVA with Holm-Sidak’s multiple comparisons test for quantification of train asynchronous release.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Transduction, Staining, Construct, Generated, Labeling

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet: ( a ) Same neuron as shown in but shown here with a larger field of view and only the PM-S7α-HaloTag/JF646 channel. Arrows point to the axon. Scale bar = 10 μm. ( b ) An example image of an axon from a neuron massively overexpressing transfected LAMP1-S7α-HaloTag (JF646) and cytosolic mRuby3. When overexpressed, this construct appears to localize to the plasma membrane, potentially confounding our analysis of this construct. ( c ) Representative image taken with the same imaging conditions as in . Neurons transduced sparsely with SYP-SYT7α-HaloTag. Arrows point to a synaptic vesicle cluster and nearby LAMP1+ structure. Scale bar = 1 μm. ( d ) Representative super-resolution optical slices of HEK293T cells expressing the location-specific SYT7α rescue constructs. From left to right, cytosolic msGFP (cyan) with PM-SYT7α (magenta), LAMP1-mRuby3 (yellow) with LAMP1-SYT7α (magenta), and SYP-mRuby3 (yellow) with SYP-SYT7α (magenta). Scale bar = 10 μm. ( e ) Average iGluSnFR ΔF/F 0 traces from SYT7 floxed strain, WT (floxed/no CRE, black, n = 13), and SYT7KO (+CRE, red, n = 13), from three independent experiments. ( f ) Fraction of active synapses (synapses releasing peak glutamate above baseline, >4 SD above noise) as a function of stimulation number during high-frequency stimulation (HFS). Values are means +/- SEM, ****p<0.0001 by two-way analysis of variance (ANOVA) comparing genotypes. ( g ) Representative anti-SYT7 immunoblot from rat cortical neurons, illustrating expression of labeled SYT7α constructs used in . ( h ) Average synchronous release percentage plotted by stimulation number, genotypes as labeled. As HFS continues, synchronous release decreases. Same data as in . Lines are third-order polynomial fits to data, for illustrative purposes only.

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Transfection, Construct, Imaging, Expressing, Western Blot, Labeling

Journal: eLife

Article Title: Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons

doi: 10.7554/eLife.67261

Figure Lengend Snippet:

Article Snippet: Briefly, we cloned SYT7 carboxy gRNA and gRNA-flanked HaloTag into pORANGE (pORANGE cloning template vector was a gift from Harold MacGillavry (Addgene plasmid # 131471; http://n2t.net/addgene :131471; RRID: Addgene_131471 )).

Techniques: Recombinant, Plasmid Preparation, Software, Sequencing