Journal: Cell reports

Article Title: Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development

doi: 10.1016/j.celrep.2022.110483

Figure Lengend Snippet: (A and D) Representative images of rat CA1 pyramidal neurons, P7, transfected in utero at E17.5 with shRNA for PlexinA3 (shPlexinA3), NP-1 (shNP-1), PlexinA4 (shPlexinA4), or PlexinD1 (shPlexinD1), or LRR, or control vector (A). Neurons co-transfected with WT-PlexinA3, PlexinA3-ΔC2 (associated Scribble; ), or PlexinA3-ΔCT (did not associate Scribble; ), or functional sGC (co-expressing sGC-α1 and -β1 subunits) (D). Neurons also co-transfected with shPlexinA3 and LRR (shPlexinA3 + LRR) (D). Neurons co-transfected with dTom marker. SO, SP, and SR layers as in . Scale bar, 100 μm. Bottom: sample tracings of neuritic arbor of representative neurons. Scale bar, 20 μm (see and ). (B and E) Quantification of average total apical dendrite length per cell for CA1 pyramidal neurons transfected as in (A) and (D) (n = 20–52 cells; data for control, shPlexinA3, and LRR were pooled among B and E). Control versus LRR or shPlexinA3 + LRR, one-way ANOVA, Dunnett’s multiple comparison test: ***p ≤*** 0.001; all other conditions, one-way ANOVA, Tukey’s multiple comparison test: ***p ≤ 0.001; ****p ≤ 0.0001. Data for shPlexinA3, shPlexinA4, and shPlexinD1 were not significantly different (ns) from control. (C and F) Quantification of average total apical dendrite branch points per cell; same dataset as in (B) and (E). Data for Control, shPlexinA3, and LRR were pooled among (C) and (F). Control versus shPlexinA3, LRR, or shPlexinA3 + LRR, one-way ANOVA, Dunnett’s multiple comparison test: **p ≤ 0.01; ***p ≤ 0.001; all other conditions, one-way ANOVA, Tukey’s multiple comparison test: **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; shPlexinA3 versus shPlexinA3 + WT-PlexinA3, Student’s t test, **p ≤ 0.01; data for shPlexinA4 or shPlexinD1 were not significantly different (ns) from control. **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Error bars represent SEM.

Article Snippet: goat anti PlexinD1 , Santa Cruz , Cat#sc-46245 (E-13).

Techniques: Transfection, In Utero, shRNA, Plasmid Preparation, Functional Assay, Expressing, Marker

Journal: Cell reports

Article Title: Semaphorin3A/PlexinA3 association with the Scribble scaffold for cGMP increase is required for apical dendrite development

doi: 10.1016/j.celrep.2022.110483

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: goat anti PlexinD1 , Santa Cruz , Cat#sc-46245 (E-13).

Techniques: Recombinant, Western Blot, Plasmid Preparation, Software

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: SH3BP1 was identified from the screen as a downstream component of Sema3E-PlexinD1 signaling, and further validated by siRNA-resistant SH3BP1 rescue and PlexinD1/SH3BP1 colocalization. (A) Automated image acquisition showed that SH3BP1 knockdown blocked Sema3E-induced collapse to similar extent as PlexinD1 siRNA. HUVECs treated with Sema3E underwent cell collapse that was inhibited by transfection with siRNA targeted specifically to PlexinD1 or SH3BP1. Bar, 100 µm. (B) Quantification of the Sema3E-induced cell collapse using the automated image analysis algorithm. ***, P < 0.0001. Error bars indicate SEM. (C) PlexinD1 and SH3BP1 colocalized at the leading edge of the cell (arrows). PlexinD1-GFP and SH3BP1-HA were coexpressed in HUVECs and stained with the corresponding antibodies. Bar, 10 µm. (D) Endogenous localization of PlexinD1 and SH3BP1 in HUVECs. PlexinD1 and SH3BP1 colocalized in the leading edge of the cells (arrows). The boxed regions are enlarged on the right. Bar, 10 µm. (E) Reintroducing SH3BP1 protein rescued SH3BP1 siRNA inhibition of Sema3E-induced collapse. HUVECs were transfected with SH3BP1 siRNA followed by transfection with control DNA or the siRNA-resistant SH3BP1 construct and treated with Sema3E or control ligand. Strong cell collapse was observed only in cells transfected with the rescue construct (arrows). Cell shape was visualized by DTAF labeling (green) and vector expression was detected by an HA antibody (red). Bar, 100 µm. (F) Quantification of the cell collapse demonstrated the ability of the siRNA-resistant SH3BP1 to fully rescue the Sema3E-induced collapse. **, P < 0.001. Error bars indicate SEM.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Transfection, Staining, Inhibition, Construct, Labeling, Plasmid Preparation, Expressing

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: Sema3E treatment causes dynamic changes in the actin cytoskeleton and cell collapse, which is inhibited by PlexinD1 or SH3BP1 knockdown. (A) Live cell imaging in control, PlexinD1, and SH3BP1 siRNA-transfected HUVECs after Sema3E treatment. Representative DIC images were taken from time-lapse videos of cells at different time points after Sema3E treatment. Control cells underwent obvious morphological changes and exhibited cell collapse in response to Sema3E treatment, whereas PlexinD1- and SH3BP1-depleted cells did not display any changes (see Videos 1 , 2 , and 3 for the entire video sequence). (B) Quantification of live imaging. Cell size at different time points was measure and normalized to time point 0. **, P < 0.001. Error bars indicate SEM. (C) Actin staining in control, PlexinD1, or SH3BP1 siRNA–transfected HUVECs treated with Sema3E at different time points. Cells transfected with the corresponding siRNA were treated with Sema3E, fixed, and stained with phalloidin (green). In untreated cells, well-organized actin networks with lamellipodia and F-actin stress fibers were seen. After 10 and 20 min of exposure to Sema3E, control cells lost their shape and saw a disruption of F-actin stress fibers. Sema3E treatment of PlexinD1 and SH3BP1 siRNA–transfected cells exhibited an organized network with lamellipodia (yellow arrows) and F-actin stress fibers (white arrows) at every time point. Blue, DAPI. Bar, 10 µm. (D) Quantification of changes in the presence of lamellipodia and stress actin fibers upon Sema3E treatment. In control cells, the number of cells with lamellipodia and stress fibers was significantly reduced after 10 and 20 min of Sema3E treatment. PlexinD1- and SH3BP1-transfected cells did not show significant changes in the presence of lamellipodia and actin stress fibers at 10 and 20 min from ligand introduction. n = 3. *, P < 0.01; **, P < 0.001. Error bars indicate SEM.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Live Cell Imaging, Transfection, Sequencing, Imaging, Staining

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: Sema3E-PlexinD1 signaling down-regulates Rac1 activity, which is medicated by SH3BP1 via its GAP activity. (A) Constitutively active Rac1 blocks Sema3E-induced collapse. Cells were transfected with a constitutively active form of Rac1 (Rac1Q61L) and treated with control ligand or Sema3E. GFP construct was cotransfected to visualize cell morphology. While the addition of Sema3E to cells overexpressing only GFP led to strong cell collapse (arrows), HUVECs transfected with Rac1Q61L were unable to undergo collapse after treatment with Sema3E. Bar, 100 µm. (B) PlexinD1 and SH3BP1 colocalized with Rac1 at the lamellipodia (arrows) of HUVECs. PlexinD1-GFP and SH3BP1-HA were cotransfected in HUVECs and stained with GFP or HA antibodies combined with a Rac1 antibody. The boxed regions are enlarged on the right. Bar, 10 µm. (C) Sema3E induced a down-regulation of Rac1 activity, which was blocked by PlexinD1 or SH3BP1 siRNA. HUVECs treated with Sema3E for 0, 2.5, 5, 10, or 20 min were lysed, precipitated with the PAK Rac1-binding domain, and blotted with a Rac1 antibody. Rac1-GTP, total Rac1, and Tubulin blots are shown. (D) Quantification of a Rac1 activity assay is shown. *, P < 0.05; **, P < 0.005. Error bars indicate SEM. (E) SH3BP1 GAP activity was required for Sema3E-induced collapse. Deletion of the GAP domain of SH3BP1 as well as the GAP activity–defective mutant failed to rescue SH3BP1 siRNA inhibition of Sema3E-induced collapse. Rescue experiments with SH3BP1ΔRhoGAP, SH3BP1-R232A, and SH3BP1-Res (full-length siRNA resistant) constructs are shown. Cell shape, DTAF labeling (green); vector expression, HA antibody (red). Arrows, cell collapse. Bar, 100 µm. (F) The results from D were quantified and the percentage of cell collapse is shown. ***, P < 0.01; n ≥ 3. Error bars indicate SEM. n.s., not significant.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Activity Assay, Transfection, Construct, Staining, Binding Assay, Mutagenesis, Inhibition, Labeling, Plasmid Preparation, Expressing

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: SH3BP1 forms a complex with PlexinD1 via its BAR domain, and Sema3E binding to PlexinD1 activates SH3BP1 by releasing it from the PlexinD1 complex. (A) Schematic illustration of the SH3BP1 constructs used in this study. The full-length SH3BP1 construct contains BAR and RhoGAP domains and SH3 motifs on the C-terminal end. In the SH3BP1ΔRhoGAP construct, the GAP domain was deleted. The SH3BP1ΔBAR construct lacked the BAR domain, and in the SH3BP1ΔSH3 construct the SH3 motif was deleted. (B) SH3BP1 is associated with PlexinD1 through the SH3BP1 BAR domain. HEK293T cells were transfected with PlexinD1-GFP and vectors expressing SH3BP1 (SH3BP1-HA or deletion constructs SH3BP1ΔBAR-HA, SH3BP1ΔRhoGAP-HA, and SH3BP1ΔSH3). Cells were lysed and immunoprecipitated with a GFP antibody and immunoblotted with an HA antibody (right). PlexinD1 and SH3BP1 expression levels in the cell lysates were determined by Western blotting with GFP and HA antibodies (left). (C) SH3BP1 dissociated from PlexinD1 upon Sema3E treatment. HEK293T cells transfected with PlexinD1-GFP and SH3BP1-HA were treated with Sema3E for 0, 10, and 20 min, then lysed and immunoprecipitated with a GFP antibody and immunoblotted with an HA antibody. The dissociation of SH3BP1 from PlexinD1 was observed 20 min after Sema3E treatment, as indicated on the right, while the protein expression levels are shown on the left.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Binding Assay, Construct, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: BAR domain deletion caused constitutive cell collapse, and Sema3E treatment enhanced the collapse. (A) Overexpression of SH3BP1ΔBAR led to changes of cell morphology in HUVECs. SH3BP1-HA, SH3BP1ΔGAP-HA, SH3BP1ΔSH3-HA, and SH3BP1ΔBAR-HA were overexpressed in HUVECs, and cells were stained with DTAF (green) and HA antibody (red). Deletion of the BAR domain caused changes in cell morphology and size compared with full-length SH3BP1 as well as GAP and SH3 deletion. Bar, 100 µm. (B) Quantification of overexpression experiments is shown. *, P < 0.01; n = 4. Error bars indicate SEM. (C) Deletion of the BAR domain of SH3BP1 partially rescued SH3BP1 siRNA inhibition of Sema3E-induced collapse. Rescue experiments with SH3BP1ΔBAR, SH3BP1ΔSH3, and SH3BP1-Res (full-length siRNA resistant) constructs are shown. Cell shape, DTAF (green); vector expression, HA antibody (red). Arrows, cell collapse. Bar, 100 µm. (D) The results from C were quantified and the percentage of cells collapsed is shown. *, P < 0.01; **, P < 0.001. n = 4. Error bars indicate SEM.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Over Expression, Staining, Inhibition, Construct, Plasmid Preparation, Expressing

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: A model of how SH3BP1–Rac1 mediates Sema3E-PlexinD1 regulation of cytoskeleton stability. (A) In the absence of Sema3E, SH3BP1 is associated with the PlexinD1 complex, and the active form of Rac1 (Rac1GTP) positively regulates actin polymerization. (B) Upon Sema3E treatment, SH3BP1 dissociates from PlexinD1, becomes activated, and through its RhoGAP domain converts GTP-Rac1 to GDP-Rac1. The decreased Rac1 activity leads to actin depolymerization and cell collapse.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Activity Assay

Journal: The Journal of Cell Biology

Article Title: An image-based RNAi screen identifies SH3BP1 as a key effector of Semaphorin 3E–PlexinD1 signaling

doi: 10.1083/jcb.201309004

Figure Lengend Snippet: PlexinD1 and SH3BP1 play a role in Sema3E induced ECs repulsion. (A) HUVECs transfected with control, PlexinD1, and SH3BP1 siRNA were grown to a confluent monolayer. Cells were photographed 16 h later. HEK293T cells expressing control ligand or Sema3E were prelabeled with Hoechst 33342 and added on top of HUVECs. HEK293T expressing Sema3E (arrows) were surrounded by a cell-free area, whereas HEK293T expressing a control ligand did not exhibit a cell-free area. PlexinD1 and SH3BP1 siRNA-transfected cells did not repel in the presence of a Sema3E source. Bright field and Hoechst 33342 channel are shown. Bar, 200 µm. (B) Quantification of cell free area surrounding HEK293T cells. *, P < 0.01; **, P < 0.001. n = 4. Error bars indicate SEM.

Article Snippet: The membranes were then incubated overnight (see next paragraph) with the following primary antibodies at 4°C: rabbit anti-GFP (Invitrogen), mouse anti-HA (Covance), mouse anti-Rac1 (BD), rabbit polyclonal antiserum raised against PlexinD1 peptide (CELVEPKKSHRQSHRKK; anti-PlexinD1 was a gift from Y. Yoshida, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ), goat anti-SH3BP1 (Everest Biotech Ltd.; ; ), rabbit VE-Cadherin (Abcam), and mouse anti-Tubulin (Sigma-Aldrich).

Techniques: Transfection, Expressing