prk5 myc rac1 t17n  (Addgene inc)

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: Recombinant DNA reagent , Rac1-DN , Addgene , Cat# 12984; RRID: Addgene_12984 , Expresses T17N variant of human myc-Rac1 under a CMV promotor.

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: Recombinant DNA reagent , Rac1-DN , Addgene , Cat# 12984; RRID: Addgene_12984 , Expresses T17N variant of human myc-Rac1 under a CMV promotor.

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: Recombinant DNA reagent , Rac1-DN , Addgene , Cat# 12984; RRID: Addgene_12984 , Expresses T17N variant of human myc-Rac1 under a CMV promotor.

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

Journal: bioRxiv

Article Title: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

doi: 10.1101/2023.04.12.536297

Figure Lengend Snippet: (A&B) Cells were transfected with plasmids encoding Rac1-WT, -T17N (dominant negative), or - Q61L (constitutively active), and treated with MPA, G, or RT as in . Cells were harvested 4 h post RT to assess Rac1 activity (A) or γ-H2AX foci by immunofluorescence (B). (C&D) Cells were transfected with indicated Rac1 plasmids (WT, constitutively active Q61L or dominant negative T17N) and irradiated with 4 Gy, followed by cell harvesting 4 h post-RT to assess p-Abi1-S323 by immunoblot. (E&F) Con-KO or Abi-KO cells were transfected with Rac1-Q61L plasmid and treated with radiation and harvested for immunoblot (E) or γ-H2AX foci IF staining (F). Data are presented as mean ± SEM from three biologically independent experiments for Figure B & F and Figure A, C, D, E are representative figures from three biologically independent experiments. Two-tailed unpaired student’s t test *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: pcDNA3.1(+)-3xFlag-tagged Abi-1 wild type and site-directed mutants (Abi-1-S323D and Abi-1-S323A) were commercially ordered from Sangon Biotech ( http://www.life-biotech.com/ ; Shanghai, China) and mutation accuracy was confirmed by sequencing. pRK5-Myc-tagged Rac1 wild type (12985), Q61L (12983), and T17N (12984) plasmids were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Activity Assay, Immunofluorescence, Irradiation, Cell Harvesting, Western Blot, Plasmid Preparation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

doi: 10.1101/2023.04.12.536297

Figure Lengend Snippet: (A) Enteroids were treated with RT (4 Gy) alone or RT (4 Gy) combined with G (50 μm) and harvested for IF staining 4 h post-RT. (B&C) Enteroids or normal human astrocytes (NHA) were treated as before and harvested for immunoblot assay. (D) NHA were transfected with wild type (WT), constitutively active (Q61L) or dominant negative (T17N) Rac1 plasmids and treated as above, and cells were harvested 4h post-RT for immunoblot. (E-G) C57BL/6J mice were treated with 7 doses of guanosine (300 mg/kg) by oral gavage or combined with one dose (10 Gy) of abdominal radiation. A subset of mice were sacrificed, and jejunums were harvested 4 h after receiving radiation for IF (E) or IHC staining (F), respectively. Another subset of mice continued to receive the rest of guanosine treatment and jejunums were harvested at day 14 for H&E (G, top panels) or ki-67 IHC staining (G, bottom panels). (H&I) Digital images of Masson’s trichrome staining for collagen deposition (blue) at ∼day 21 (H) and lung hydroxyproline content was quantified (I). (J) A schematic summary of our study.

Article Snippet: pcDNA3.1(+)-3xFlag-tagged Abi-1 wild type and site-directed mutants (Abi-1-S323D and Abi-1-S323A) were commercially ordered from Sangon Biotech ( http://www.life-biotech.com/ ; Shanghai, China) and mutation accuracy was confirmed by sequencing. pRK5-Myc-tagged Rac1 wild type (12985), Q61L (12983), and T17N (12984) plasmids were obtained from Addgene.

Techniques: Staining, Western Blot, Transfection, Dominant Negative Mutation, Immunohistochemistry