polyclonal rabbit anti-homer1 antibody  (Synaptic Systems)

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic representation of functional domains of mDia1. Rho-binding domain (RBD), Diaphanous inhibitory domain (DID), Dimerization domain (DD), Coiled coil domain (CC), Formin homology domain 1 (FH1), Formin homology domain 2 (FH2), Diaphanous autoinhibitory domain (DAD). The unstructured N-terminus (first 60 amino acids) contains three basic stretches and was truncated in the ΔN mutant. ( B ) Endocytic decay constants of Synaptophysin-pHluorin traces from hippocampal neurons transfected with shRNAmiR against no mammalian target ( shCTRmiR ) or Diaph1 ( shmDia1miR ) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transfected with plasmids encoding mDia1-WT-mCherry (τ shmDia1miR + mDia1-WT =20.0±0.8 s), mDia1-ΔN-mCherry (τ shmDia1miR + mDia1-ΔN =34.5±2.9 s), or mCherry alone (τ shCTRmiR = 21.8±1.1 s, τ shmDia1miR = 30.4±1.9 s) to exclude artifacts from overexpression (p shCTRmiR vs shmDia1miR < 0.05; p shmDia1miR vs shmDia1miR + mDia1-WT <0.01; p shmDia1miR + mDia1-WT vs shmDia1miR + mDia1-ΔN <0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=5 independent experiments from n shCTRmiR = 41 videos, n shmDia1miR = 51 videos, n shmDia1miR + mDia1-WT =35 videos, n shmDia1miR + mDia1-ΔN =37 videos. ( C ) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 μM Dynasore for 10 min before fixation and immunostained for Bassoon (presynaptic marker, magenta), mDia1 (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. ( D ) Averaged normalized line profiles for synaptic distribution of mDia1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=235 synapses. ( E ) Volcano plot of proteins associating with synaptic mDia1 analyzed by label-free proteomics of anti-mDia1 versus control (CTR) immunoprecipitates from detergent-extracted mouse synaptosomes (P2’ fraction). The logarithmic ratios of protein intensities are plotted against negative logarithmic p-values derived from a two-tailed student’s t-test. N=3 independent experiments. Each dot represents one protein. Selected cytoskeletal hits include: Actin, Myosin IIB (MyoIIB), and Rac1. Selected endocytic hits include Amphiphysin (p<0.05), Dynamin1, Endophilin-A1, PACSIN1, PACSIN2 (p<0.05), and Synaptojanin1. ( F ) Endogenous immunoprecipitation of mDia1 from detergent-extracted mouse synaptosomes (P2’ fraction) using mDia1-specific antibodies. Immunoprecipitates were analyzed by immunoblotting for mDia1, Dynamin1, and β-Actin. ( G ) Representative three-channel time-gated STED images of synapses from hippocampal cultures transduced with wild-type Dynamin1 (WT) or GTPase-deficient Dynamin1 (K44A) in response to 200 AP (40 Hz, 5 s) stimulation. Cells were immunostained for Bassoon (magenta), mDia1 (cyan), and Homer1 (green). Scale bar, 250 nm. ( H ) Presynaptic mDia1 levels in synapses treated with 0.1% DMSO (100±7.3) or 80 µM Dynasore (145.8±9.3; p=0.0001; one sample Wilcoxon test) for 10 min in response to 200 AP (40 Hz, 5 s) stimulation. Absolute line profiles of mDia1 overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 92 synapses, n Dynasore = 135 synapses. ( I ) Presynaptic mDia1 levels in synapses from hippocampal neurons transduced with wild-type Dynamin1 (WT; 100±6.2) or GTPase-deficient Dynamin1 (K44A; 142.9±8.3, p<0.0001, one sample Wilcoxon test) in response to 200 AP (40 Hz, 5 s) stimulation. Line profiles of mDia1 overlapping with Bassoon distribution were integrated. Data shown are normalized to Dynamin1-WT (set to 100) and expressed as mean ± SEM. N=2 independent experiments from n WT = 43 synapses, n K44A = 51 synapses. Figure 3—source data 1. Numerical source data for . Figure 3—source data 2. Original scan for the anti-mDia1 and anti-Dynamin1 immunoblots from . Figure 3—source data 3. Original scan for the anti-Actin immunoblot from . Figure 3—source data 4. Original scans for immunoblots from with highlighted bands and sample labels.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Functional Assay, Binding Assay, Mutagenesis, Transfection, Over Expression, Marker, Control, Derivative Assay, Two Tailed Test, Immunoprecipitation, Western Blot, Transduction

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Membrane levels of mDia1-WT-mCherry versus mDia1-ΔN-mCherry proteins overexpressed in HEK293T cells. Membrane and cytosolic cellular fractions were isolated by ultracentrifugation and analyzed by immunoblotting with specific antibodies (LAMP1) and in-gel fluorescence of mCherry tags. ( B ) Densitometric quantification of mDia1-WT versus mDia1-ΔN (0.6±0.1; p<0.05, one sample t-test) membrane-associated protein levels. Data shown are normalized to mDia1-WT (set to 1) and expressed as mean ± SEM. Representative immunoblot is shown in A. N=5 independent experiments. ( C ) Averaged normalized Synaptophysin-pHluorin fluorescence from stimulated (200 action potentials (APs), 40 Hz, 5 s) hippocampal neurons transfected with shCTRmiR or shmDia1miR . For rescue experiments, neurons were co-transfected with plasmids encoding mDia1-WT-mCherry, mDia1-ΔN-mCherry or mCherry alone (shCTRmiR & shmDia1miR). Endocytic decay constants are shown in . ( D ) Full volcano plot of proteins from associating with synaptic mDia1 analyzed by label-free proteomics of anti-mDia1 versus control (CTR) immunoprecipitates from detergent-extracted mouse synaptosomes (P2’ fraction). The cyan dot shows the specific enrichment of mDia1 as the bait protein of the immunoprecipitation (p<0.001, two-tailed student’s t-test). N=3 independent experiments. ( E ) Endogenous co-immunoprecipitation of Myosin IIB by mDia1 from detergent-extracted mouse synaptosomes (P2’ fraction). Samples were analyzed by immunoblotting using specific antibodies against mDia1, Myosin IIB (MyoIIB), and β-Actin. ( F ) Representative three-channel time-gated STED image of a synapse from hippocampal cultures fixed and immunostained for Bassoon (presynaptic marker, magenta), Myosin IIB (cyan), and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. ( G ) Averaged normalized line profiles for synaptic distribution of Myosin IIB and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=3 independent experiments from n=267 synapses. ( H ) Rationale for quantification of presynaptic protein levels of interest. The presynapse was defined by the normalized Bassoon distribution (purple fraction, cut off at the cross-section with the Homer1 profile), and corresponding absolute individual synaptic line profiles were integrated. ( I ) Postsynaptic F-Actin levels in synapses treated with 0.1% dimethyl sulfoxide (DMSO) (42.6±3.4) or 80 µM Dynasore (56.5±3.3; p<0.001, Mann-Whitney test) for 10 min before fixation from . Data shown are normalized to presynaptic DMSO values from (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 92 synapses, n Dynasore = 135 synapses. ( J ) Quantification of Bassoon and Homer1 levels in synapses treated with 0.1% DMSO (100.0±4.5 for Bassoon; 100.0±4.3 for Homer1) or 80 µM Dynasore (103.7±4.1 for Bassoon; 98.6±5.3) for 10 min before fixation. Data shown are normalized to DMSO values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 132 synapses, n Dynasore = 128 synapses. Figure 3—figure supplement 1—source data 1. Original scan for in-gel mCherry fluorescence from . Figure 3—figure supplement 1—source data 2. Original scan for the anti-LAMP1 immunoblot from . Figure 3—figure supplement 1—source data 3. Original scans from with highlighted bands and sample labels. Figure 3—figure supplement 1—source data 4. Numerical source data of , C, D, G, I, J. Figure 3—figure supplement 1—source data 5. Original scans for mCherry fluorescence in gels used for analysis are shown in . Figure 3—figure supplement 1—source data 6. Original scans for mCherry fluorescence in gels used for analysis are shown in with highlighted bands and sample labels. Figure 3—figure supplement 1—source data 7. Original scan for the anti-mDia1 immunoblot from . Figure 3—figure supplement 1—source data 8. Original scan for the anti-Myosin IIB immunoblot from . Figure 3—figure supplement 1—source data 9. Original scan for the anti-Actin immunoblot from . Figure 3—figure supplement 1—source data 10. Original scans for immunoblots in with highlighted bands and sample labels.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Membrane, Isolation, Western Blot, Fluorescence, Transfection, Control, Immunoprecipitation, Two Tailed Test, Marker, MANN-WHITNEY

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures transduced with shCTR or shmDia1 +3 , targeting Diaph1/2 genes, fixed and immunostained for Bassoon (presynaptic marker, magenta), F-Actin (cyan) and Homer1 (postsynaptic marker, green). Scale bar, 250 nm. ( B ) Averaged normalized line profiles for synaptic distribution of F-Actin and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=4 independent experiments from n=154 synapses. ( C ) Presynaptic F-Actin levels in synapses treated with 0.1% dimethyl sulfoxide (DMSO) (100±4.8) or 80 µM Dynasore (134.7±6.8; p=0.001, one sample Wilcoxon test) for 10 min before fixation (Representative images in ). Cells were immunostained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Absolute line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n DMSO = 207 synapses, n Dynasore = 211 synapses. ( D ) Endocytic decay constants of vesicular glutamate transporter 1 (vGLUT1)-pHluorin traces from hippocampal neurons transduced with lentiviral particles encoding shCTR (τ shCTR = 20.7 ± 0.9 s) or shmDia1 (τ shmDia1 = 26.4±2.0 s) in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue experiments, neurons were co-transduced with lentiviruses encoding mDia1-WT-SNAP (τ shmDia1 + mDia1-WT =16.1±1.9 s) or mDia1-K994A-SNAP (τ shmDia1 + mDia1-K994A =29.0±1.9 s) (p shmDia1 vs shmDia1 + mDia1-WT <0.01; p shmDia1 + mDia1-WT vs shmDia1 + mDia1-K994A <0.001, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR = 21 videos; n shmDia1 =21 videos, n shmDia1 + mDia1-WT =16 videos, n shmDia1 + mDia1-K994A =19 videos. ( E ) Presynaptic F-Actin levels in synapses from hippocampal cultures transduced with shCTR (100±6.4) or shmDia1 +3 (58.1±2.9; p<0.001, one sample Wilcoxon test). Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM. N=4 independent experiments from n shCTR = 155 synapses, n shmDia1+3 = 158 synapses. ( F ) Representative confocal and two-channel time-gated STED images of endogenous β-Actin (cyan) in vGLUT1 (magenta) positive boutons from hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 . Scale bar, 250 nm. ( G ) Analysis of presynaptic endogenous β-Actin levels in vGLUT1 positive boutons from shCTR (100±6.3) and shmDia1 +3 (47.7±4.3; p<0.0001 one-sample Wilcoxon test) transduced neurons. β-Actin STED mean intensity was measured using a confocal vGLUT1 signal as a mask. Data shown are normalized to shCTR (set to 100) and expressed as mean ± SEM from two independent experiments and n shCTR = 67 synapses, n shmDia1+3 = 53 synapses. ( H ) Endocytic decay constants of vGLUT1-pHluorin traces for neurons transduced with shCTR or shmDia1 +3 in response to 40 AP (20 Hz, 2 s) stimulation. Neurons were pre-incubated with 0.1% DMSO or 1 µM Jasplakinolide (Jasp) for 30 min in the media before imaging (τ shCTR + DMSO = 13.4±1.0 s, τ shCTR + Jasp = 15.0±2.2 s, τ shmDia1+3 + DMSO =25.0±2.7 s, τ shmDia1+3 + Jasp =15.6±2.4 s; p shCTR vs shmDia1+3 < 0.01; p shmDia1+3 + DMSO vs shmDia1+3 + Jasp <0.05, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 32 videos, n shmDia1+3 + DMSO =35 videos, n shCTR + Jasp = 33 videos; n shmDia1+3 + Jasp =34 videos. Figure 4—source data 1. Numerical source data for .

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Transduction, Marker, Incubation, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Representative three-channel time-gated stimulated emission depletion (STED) images of synapses from hippocampal cultures treated with 0.1% dimethyl sulfoxide (DMSO) or 80 µM Dynasore for 10 min. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. Corresponding analysis of presynaptic F-Actin levels is shown in . ( B ) Representative three-channel time-gated STED images of synapses from hippocampal cultures transduced with Dynamin1-WT or Dynamin1-K44A. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. ( C ) Presynaptic F-Actin levels in synapses from neurons transduced with Dynamin1-WT (100±5.9) or Dynamin1-K44A (119.8±6.2, p<0.01, one sample t-test) in B. Absolute line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to WT (set to 100) and expressed as mean ± SEM. n WT = 54 synapses, n K44A = 49 synapses. ( D ) Averaged normalized vesicular glutamate transporter 1 (vGLUT1)-pHluorin fluorescence traces for neurons transduced with shCTR or shmDia1 in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. For rescue purposes, cells were co-transduced with mDia1-WT-SNAP or mDia1-K994A-SNAP. Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR = 21 videos; n shmDia1 =21 videos; n shmDia1 + mDia1-WT =16 videos; n shmDia1 + mDia1-K994A =19 videos. Corresponding endocytic decay constants are shown in . ( E ) Postsynaptic F-Actin levels in synapses transduced with shCTR (100.0±6.4) or shmDia1 +3 (89.3±6.4) from . Data shown are normalized to shCTR values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n shCTR = 206 synapses, n shmDia1+3 = 135 synapses. ( F ) Quantification of Bassoon and Homer1 levels in synapses transduced with shCTR (100.0±4.7 for Bassoon; 100.0±4.5 for Homer1) or shmDia1 +3 (101.4±4.8 for Bassoon; 92.4±4.0). Data shown are normalized to DMSO values (set to 100) and expressed as mean ± SEM. N=3 independent experiments from n shCTR = 158 synapses and n shmDia1+3 = 159 synapses. ( G ) Representative STED images of endogenous β-Actin in vGLUT1 positive synapses in hippocampal neurons transduced with shCTR or shmDia1 +3 and treated with 0.1% DMSO or 1 µM Jasplakinolide for 45 min. Neurons were co-transfected with pOrange-GFP-β-Actin knock-in and vGLUT1-mCherry plasmids before fixation and immunostaining. Scale bar, 2.5 µm. ( H ) Averaged normalized vGLUT1-pHluorin fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 40 AP (20 Hz, 2 s) stimulation. Neurons were pre-incubated with 0.1% DMSO or 1 µM Jasplakinolide (Jasp) for 30 min in the cell media before imaging. Data are expressed as mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 32 videos, n shmDia1+3 + DMSO =35 videos, n shCTR + Jasp = 33 videos; n shmDia1+3 + Jasp =34 videos. The corresponding endocytic decay constants are shown in . Figure 4—figure supplement 1—source data 1. Numerical source data of , D, E, F, H.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Staining, Transduction, Fluorescence, Transfection, Knock-In, Immunostaining, Incubation, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic representation of activation of mDia1 by RhoA-GTP and positive feedback loop of mDia1 on RhoA-GTP levels through GEF stimulation. ( B ) Representative three-channel time-gated STED image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), RhoA (cyan), and Homer1 (green). Scale bar, 250 nm. ( C ) Averaged normalized line profiles for synaptic distribution of RhoA and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM. N=5 independent experiments from n=230 synapses. ( D ) Endocytic decay constants of averaged normalized Synaptophysin-pHluorin fluorescence traces in response to 200 action potential (AP) (40 Hz, 5 s) stimulation. Neurons were transfected with the annotated combinations of plasmids encoding wild-type (WT) or dominant-negative (DN, T19N mutation) RhoA and RhoB (τ RhoA-WT + RhoB-WT =18.4±0.7 s, τ RhoA-WT + RhoB-DN = 16.0±1.0 s, τ RhoA-DN + RhoB-WT =19.8±2.4 s, τ RhoA-DN + RhoB-DN =30.1±1.0 s; p RhoA-WT + RhoB-WT vs RhoA-DN + RhoB-DN <0.01, one-way ANOVA with Tukey’s post-test). Data shown represent mean ± SEM. N=3 independent experiments from n RhoA-WT + RhoB-WT =21 videos, n RhoA-DN + RhoB-WT =31 videos, n RhoA-WT + RhoB-DN =23 videos, n RhoA-DN + RhoB-DN =22 videos. ( E ) Analysis of RhoA activity by RhoA-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 using immobilized Rhotekin as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, RhoA, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. ( F ) Densitometric quantification of RhoA-GTP normalized to total RhoA levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (0.7±0.0, p<0.001, one sample t-test) from immunoblots exemplified in E. Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. Figure 5—source data 1. Numerical source data for . Figure 5—source data 2. Original scans for the anti-mDia1, anti-Tubulin, anti-RhoA, and anti-mDia3 immunoblots from . Figure 5—source data 3. Original scans for immunoblots from with highlighted bands and sample labels. Figure 5—source data 4. Original scans for the anti-RhoA immunoblots used for analysis are shown in . Figure 5—source data 5. Original scans for immunoblots used for analysis are shown in with highlighted bands and sample labels.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Activation Assay, Fluorescence, Transfection, Dominant Negative Mutation, Mutagenesis, Activity Assay, Expressing, Western Blot, Transduction

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Schematic of the interplay between RhoA and Rac1 signaling via GTPase regulatory proteins (e.g. GTPase activating proteins (GAPs) among others) common for RhoA and Rac1. ( B ) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal neurons expressing shCTR or shmDia1 +3 utilizing immobilized PAK as a bait. Samples were analyzed by immunoblotting for mDia1, mDia3, Rac1, and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. ( C ) Densitometric quantification of Rac1-GTP normalized to total Rac1 levels (input) in lysates from neurons transduced with shCTR or shmDia1 +3 (2.2±0.2; p<0.05, one sample t-test) from immunoblots exemplified in ( B ). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. ( D ) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal cultures, fixed and immunostained for Bassoon (magenta), Rac1 (cyan), and Homer1 (green). Scale bar, 250 nm. ( E ) Averaged normalized line profiles for synaptic distribution of Rac1 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data represent mean ± SEM. N=3 independent experiments from n=79 synapses. ( F ) Averaged normalized vGAT-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) in the imaging buffer. Data shown represent the mean ± SEM. N=8 independent experiments from n shCTR + DMSO = 46 videos, n shmDia1+3 + DMSO = 45 videos, n shCTR + EHT 1864 = 42 videos, n shmDia1+3 + EHT 1864 = 43 videos. ( G ) Endocytic decay constants of vGAT-CypHer traces in F: τ shCTR + DMSO = 14.7±0.9 s, τ shmDia1+3 + DMSO =27.5±2.3 s, τ shCTR + EHT 1864 = 30.3±6.7 s, τ shmDia1+3 + EHT 1864 = 41.0±4.3 s; p shCTR + DMSO vs shmDia1+3 + DMSO <0.05, p shCTR + DMSO vs shmDia1+3 + EHT 1864 < 0.0001, Kruskal-Wallis test with Dunn’s post-test. Data represent mean ± SEM. ( H ) Endocytic decay constants of Synaptophysin-pHluorin traces of neurons transduced with shCTR (τ shCTR = 12.0±0.7 s) or shmDia1 +3 (τ shmDia1+3 = 22.7±2.0 s) and transfected with constitutively active Rac1 (Rac1-CA; Q61L variant; τ shCTR + Rac1-CA =13.6±1.2 s, τ shmDia1+3 + Rac1-CA =13.3±1.4 s) or dominant negative Rac1 (Rac1-DN; T17N variant; τ shCTR + Rac1-DN = 27.8±1.3 s, τ shmDia1+3 + Rac1-DN = 33.4±1.6 s) in response to 200 AP (40 Hz, 5 s) stimulation (p shCTR vs shmDia1+3 < 0.01; p shCTR vs shCTR + Rac1-DN <0.0001, p shCTR vs shmDia1+3 + Rac1-DN <0.01, p shmDia1+3 vs shmDia1+3 + Rac1-DN <0.01, one-way ANOVA with Tukey’s post-test). Data are expressed as mean ± SEM. N=3 independent experiments from n shCTR = 12 videos, n shmDia1+3 = 23 videos; n shCTR + Rac1-CA =10 videos, n shmDia1+3 + Rac1-CA =14 videos, n shCTR + Rac1-DN = 9 videos; n shmDia1+3 + Rac1-DN = 13 videos. Figure 6—source data 1. Original scans for the anti-mDia3, anti-Tubulin, and anti-Rac1 immunoblots from . Figure 6—source data 2. Original scan for the anti-mDia1 immunoblot from . Figure 6—source data 3. Original scans for immunoblots from with highlighted bands and sample labels. Figure 6—source data 4. Numerical source data for . Figure 6—source data 5. Original scans for the anti-Rac1 immunoblots used for analysis are shown in . Figure 6—source data 6. Original scans for immunoblots used for analysis are shown in with highlighted bands and sample labels.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Activity Assay, Expressing, Western Blot, Transduction, Fluorescence, Imaging, Transfection, Variant Assay, Dominant Negative Mutation

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet: ( A ) Analysis of Rac1 activity by Rac1-GTP pulldown (PD) from whole-cell lysates (input) of mouse hippocampal cultures upon inhibition of Rho activity utilizing immobilized PAK as bait. Cells were treated with 0.1% DMSO or 10 µM Rho Inhibitor (Rhosin) for 2 hr before harvest. Samples were analyzed by immunoblotting for Rac1 and Tubulin using specific antibodies. Input, 10% of material used for the pulldown. The contrast of pulldown and input blots was seperately adjusted for visualization purposes. ( B ) Representative three-channel time-gated STED images of synapses from hippocampal cultures treated with 0.1% DMSO or 10 µM Rac1 Inhibitor (EHT 1864) for 2 hr. Cells were fixed and stained for Bassoon (magenta), F-Actin (cyan), and Homer1 (green). Scale bar, 250 nm. ( C ) Presynaptic F-Actin levels in synapses of neurons treated with 0.1% DMSO (100±8.5) or 10 µM Rac1 Inhibitor (EHT 1864; 58.6±6.5; p<0.0001, one sample Wilcoxon test) for 2 hr. Line profiles of F-Actin overlapping with Bassoon (presynapse) distribution were integrated. Data shown are normalized to DMSO (set to 100) and expressed as mean ± SEM. n DMSO = 30, n EHT 1864 = 46 from two independent experiments. ( D ) Minima of background-corrected vGAT-CypHer fluorescence traces (surface normalized) for neurons treated with 0.1% DMSO (1.0±0.2 for shmDia1 +3 ) or 10 µM Rac1 Inhibitor (EHT 1864; 0.8±0.1 for shCTR ; 0.8±0.1 for shmDia1 +3 ) in response to 200 AP stimulation (40 Hz, 5 s). Data represent mean ± SEM. Values were normalized to DMSO-treated shCTR (set to 1). N=8 independent experiments from n shCTR + DMSO = 46 videos, n shmDia1+3 + DMSO =45 videos, n shCTR + EHT 1864 = 42 videos, n shmDia1+3 + EHT 1864 = 43 videos. ( E ) Averaged normalized Synaptophysin-pHluorin fluorescence traces from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR or shmDia1 +3 and transfected with plasmids for expression of constitutively-active Rac1 (Rac1-CA; Q61L variant) or dominant-negative Rac1 (Rac1-DN; T17N variant). Data represent mean ± SEM. N=3 independent experiments from n shCTR = 12 videos, n shmDia1+3 = 23 videos, n shCTR + Rac1-CA =10 videos, n shmDia1+3 + Rac1-CA =14 videos, n shCTR + Rac1-DN = 9 videos; n shmDia1+3 + Rac1-DN = 13 videos. The corresponding endocytic decay constants are shown in . ( F ) Maxima of background-corrected Synaptophysin-pHluorin fluorescence traces (surface normalized maximum values of traces shown in E) from stimulated (200 APs; 40 Hz, 5 s) hippocampal neurons transduced with lentiviruses encoding shCTR (F max /F 0 =1.3±0.0) or shmDia1 +3 (F max /F 0 =1.5±0.0) and transfected with plasmids encoding CA (F max /F 0 shCTR + Rac1-CA =1.4±0.2; F max /F 0 shmDia1+3 + Rac1-CA =1.5±0.1) or DN versions (F max /F 0 shCTR + Rac1-DN = 1.2±0.1; F max /F 0 shmDia1+3 + Rac1-DN = 1.3±0.1) of Rac1. Data represent mean ± SEM. ( G ) Densitometric quantification of Cdc42-GTP normalized to total Cdc42 levels in lysates from shmDia1 +3 transduced neurons (2.7±0.6; p<0.05, one sample t-test). Values for shCTR were set to 1. Data are expressed as mean ± SEM from N=3 independent experiments. ( H ) Representative three-channel time-gated stimulated emission depletion (STED) image of synapses from hippocampal mouse cultures, fixed and immunostained for Bassoon (magenta), Cdc42 (cyan), and Homer1 (green). Scale bar, 250 nm. ( I ) Averaged normalized line profiles for synaptic distribution of Cdc42 and Homer1 relative to Bassoon (Maximum set to 0 nm). Data are expressed as mean ± SEM (N=3; n=96 synapses). ( J ) Averaged normalized vesicular glutamate transporter 1 (vGAT)-CypHer fluorescence traces for neurons transduced with shCTR or shmDia1 +3 in response to 200 AP (40 Hz, 5 s) stimulation. Cells were acutely treated with 0.1% DMSO or 10 µM Cdc42 Inhibitor (ML141) in the imaging buffer. Data shown represent the mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 31 videos, n shmDia1+3 + DMSO =33 videos, n shmDia1+3 + ML141 =32 videos. ( K ) Endocytic decay constants of vGAT-CypHer traces in J: τ shCTR + DMSO = 15.6±1.0 s, τ shmDia1+3 + DMSO =28.0±3.1 s, τ shCTR + ML141 =17.6±1.6 s, τ shmDia1+3 + ML141 =33.1 ± 7.7 s; p shCTR + DMSO vs shmDia1+3 + DMSO <0.01, Kruskal-Wallis test with Dunn’s post-test. Data shown represent the mean ± SEM. N=6 independent experiments from n shCTR + DMSO = 31 videos, n shmDia1+3 + DMSO =33 videos, n shCTR + ML141 =29 videos, n shmDia1+3 + ML141 =32 videos. Figure 6—figure supplement 1—source data 1. Original scan for the anti-Rac1 immunoblots from . Figure 6—figure supplement 1—source data 2. Original scan for the anti-Tubulin immunoblot from . Figure 6—figure supplement 1—source data 3. Original scans for immunoblots in with highlighted bands and sample labels. Figure 6—figure supplement 1—source data 4. Numerical source data of , D, E, F, G, I, J, K. Figure 6—figure supplement 1—source data 5. Original scans for anti-Cdc42 immunoblots used for analysis are shown in . Figure 6—figure supplement 1—source data 6. Original scans for anti-Cdc42 immunoblots used for analysis are shown in with highlighted bands and sample labels.

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Activity Assay, Inhibition, Western Blot, Staining, Fluorescence, Transduction, Transfection, Expressing, Variant Assay, Dominant Negative Mutation, Imaging

Journal: eLife

Article Title: Rho GTPase signaling and mDia facilitate endocytosis via presynaptic actin

doi: 10.7554/eLife.92755

Figure Lengend Snippet:

Article Snippet: Antibody , Homer1 (rabbit polyclonal) , Synaptic Systems , Cat# 160 003; RRID: AB_887730 , IC (1:200).

Techniques: Knock-Out, Control, Recombinant, Plasmid Preparation, Variant Assay, Transfection, shRNA, Binding Assay, Mutagenesis, Software, Sequencing

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-Homer1 , Synaptic Systems , Cat# 160 003, RRID: AB_887730 , 1:1,000 for IF.

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

Journal: Cell reports

Article Title: Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke

doi: 10.1016/j.celrep.2022.111137

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Homer1 polyclonal antibody , Sigma-Aldrich , Cat#ABN37, RRID:AB_11214387.

Techniques: Plasmid Preparation, Recombinant, Software