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

Abstract

Extracellular signals have to be precisely interpreted intracellularly and translated into diverse cellular behaviors often mediated by cytoskeletal changes. Semaphorins are one of the largest families of guidance cues and play a critical role in many systems. However, how different cell types translate extracellular semaphorin binding into intracellular signaling remains unclear. Here we developed and performed a novel image-based genome-wide functional RNAi screen for downstream signaling molecules that convert the interaction between Semaphorin 3E (Sema3E) and PlexinD1 into cellular behaviors. One of the genes identified in this screen is a RhoGAP protein, SH3-domain binding protein 1 (SH3BP1). We demonstrate that SH3BP1 mediates Sema3E-induced cell collapse through interaction with PlexinD1 and regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) activity. The identification and characterization of SH3BP1 as a novel downstream effector of Sema3E-PlexinD1 provides an explanation for how extracellular signals are translated into cytoskeletal changes and unique cell behavior, but also lays the foundation for characterizing other genes identified from our screen to obtain a more complete picture of plexin signaling.

Figure 1.

An image-based genome-wide screen to unbiasedly identify Sema3E-PlexinD1 downstream signaling molecules. (A) Schematic illustration of the screen strategy. HUVECs endogenously express PlexinD1 and undergo cell collapse after Sema3E treatment. The RNAi screen identified genes that, when knocked down, block the Sema3E-induced cell collapse. (B) Schematic illustration of the automated screen procedure. Cells were transfected with smart pools of siRNA (four different sequence targets for each gene) for each well of the 384-well plates, and after 48 h Sema3E was added to the culture media for 25 min. Cells were fixed, stained, and imaged, and the cell collapse phenotype was quantified using the combination of CellProfiler and our custom-developed image analysis algorithm. (C) Automated imaging of a positive control well (PlexinD1 siRNA-transfected) and a negative control well (nontargeting siRNA-transfected) for each 384-well screening plate (images were generated from the DiI channel). HUVECs were transfected with nontargeting siRNA or PlexinD1 siRNA and treated with control ligand or Sema3E. Sema3E caused cell collapse resulting in decreased surface area and the appearance of protrusions (arrows). These cytoskeletal changes were completely blocked by PlexinD1 siRNA. Bar, 100 µm. (D) Quantification of cell collapse. Unlike cells treated with the control ligand, cells stimulated with Sema3E underwent cytoskeletal collapse, which was significantly blocked when PlexinD1 siRNA was used. Error bars indicate SD. *, P < 0.01. Control nontargeting siRNA did not alter the cellular response elicited by Sema3E.

Figure 2.

Automated image analysis of HUVEC morphology and Sema3E-induced cell collapse. (A) Raw images of cells acquired from a well of a screening plate by automated microscopy. DiI labeling combined with image analysis algorithms enabled automated quantification of cellular morphological changes at the screening end point. (B) Cellular membrane boundaries were overlaid on top of the raw image so that cell surface area could be automatically measured. (C) Cell protrusions were labeled in blue on top of the segmented cell masks so that the number of protrusions per cell could be counted. The green regions were the bumps on the cell surface that were too short to be classified as protrusions. Bar, 100 µm. (D) Physical parameters used to classify collapsed cells were determined based on the cell surface area distribution from the control wells (top, histogram) in combination with cell protrusion counts. Cells were classified as collapsed based on a reduction in their surface area and/or the presence of protrusions. (E) siRNAs that blocked Sema3E-induced HUVEC collapse were classified as strong hits if the percentage of cells collapsed was 3 SDs away from the plate-matched negative controls and within 3 SDs of the plate-matched positive controls (PlexinD1 siRNA; red box). siRNAs that partially blocked collapse were classified as weak hits (blue box).

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.

Figure 8.

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.

Figure 9.

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.