Journal: iScience

Article Title: Large-scale analysis of cell-cell communication reveals angiogenin-dependent tumor progression in clear cell renal cell carcinoma

doi: 10.1016/j.isci.2023.108367

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Article Snippet: PE anti-human Plexin-B2 (Clone REA626) , Miltenyi , Cat# 130-126-532; RRID: AB_2889732.

Techniques: Control, Recombinant, Transfection, Luminex, Software, FCAP Assay

Journal: Nature communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2.

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: Fig. 3 | Plexin-B2 affects membrane internalization in GSCs. a Dextran uptake assay of SD2 GSCs labeled with SPY-Actin. Enlarged images of boxed areas are shown below. Box plots show areas of dextran+ puncta per cell, with 25–75% quartiles, median (line), and mean (plus sign). n = 85 cells for Ctrl, n = 44 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two-sided. b Top, live cell confocal plane images of SD2 GSCs with side views of z-stacks show intracellular localization of diffuse dextran- Alexa 488 in PB2 KO cells, whereas Ctrl cells contained only dextran+ endocytic vesicles. Bottom, histograms of fluorescence profiles show bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n = 177 cells for Ctrl, n = 161 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two- sided. c, d Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell images at 72 hr after transfection show fluorescent probes on

Article Snippet: Doxycycline-inducible Plexin-B2 knockdown or overexpression Temporally controlled knockdownofPlexin-B2wasachievedwithTETON lentiviral vectors expressing doxycycline (Dox)-inducible shRNA targeting PLXNB2 (pLKO-Tet-On-PLXNB2–shRNA2; deposited as Addgene #98400).

Techniques: Membrane, Labeling, MANN-WHITNEY, Transfection

Journal: Nature communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2.

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: Fig. 6 | Plexin-B2 KO affects PIP2 localization and inner membrane surface charge in GSCs. a Left, schematic of PH(PLCD1)-GFP PIP2 probe. Right, live-cell imaging at 72 hr post transfection reveals that the PH(PLCD1)-GFP probes were largely internalized in control GSCs (arrow), but retained on membrane of PB2 KO GSCs (arrowhead). b Left, still images from videography show accumulation of PH(PLCD1)-GFP probes (arrow) in front of the nucleus (NucSpot) of migrating control SD2 in tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Dashed lines delineate cell boundary. Long arrow denotes direction of migration. Right, quantifications of the ratio of PH(PLCD1)-GFP fluorescence intensity at front vs. rear of GSCs during passage. For 3 µm: n = 15 cells for WT, n = 13 cells for PB2 KO. For 8 µm: n = 13 cells for WT, n = 16 cells for PB2 KO. One-way ANOVA followed by

Article Snippet: Doxycycline-inducible Plexin-B2 knockdown or overexpression Temporally controlled knockdownofPlexin-B2wasachievedwithTETON lentiviral vectors expressing doxycycline (Dox)-inducible shRNA targeting PLXNB2 (pLKO-Tet-On-PLXNB2–shRNA2; deposited as Addgene #98400).

Techniques: Membrane, Live Cell Imaging, Transfection, Control, Migration

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Schematic of CRISPR/Cas9-mediated PLXNB2 knockout (KO) with small guide (sg) RNA targeting second coding exon. b Western blots show Plexin-B2 expression in SD2 GSCs, with β-actin as loading control. Note Plexin-B2 precursor at 240 kDa and mature form at 170 kDa. c IF images show Plexin-B2 expression in SD2 GSCs, with Hoechst nuclear counterstain. d Left, schematic of atomic force microscopy (AFM) indentation method to probe cell stiffness by cantilever deflection. Middle, AFM indentation curves of SD2; right, box plots of cell stiffness, showing 25–75% quartiles, median (line), and mean (plus sign). n = 6 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. e Left, depiction of membrane tension measurement with optical tweezers. Middle, force measurements during tether extrusion (shaded box). Right, quantifications of tether extrusion forces. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 5 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. f Left, schematic of FLIM of cell membranes labeled with Flipper-TR membrane dye, with low and high membrane tension associated with shorter and longer lifetimes, respectively (figure modified after ref. ). Middle top, representative FLIM images, with lifetime heatmap shown on right. Middle bottom, images show similar fluorescence intensities of Flipper-TR dye in Ctrl and PB2 KO cells. Right top, violin plots show fluorescence lifetime from 3 images per group. Two-sided unpaired t-test. Right bottom, phasor plots of FLIM image data, with arrow indicating a shift to shorter lifetime values for PB2 KO cells. g Live-cell imaging of SD3 GSCs labeled with SPY-actin show differences of cortical F-actin (arrows) between Ctrl and PB2 KO GSCs. NucSpot for nuclear staining. Right, box plots of SPY-actin cortical intensity, with 25–75% quartiles, median (line), and mean (plus sign). For Ctrl, n = 37 cell cortical areas; for PB2 KO, n = 28 cell cortical areas. Two-sided unpaired t-test. h Model of Plexin-B2 regulation of cortical contractility and membrane tension. Source data are provided as a Source Data file.

Article Snippet: The plasmids for lenti- PLXNB2 expression have been deposited at Addgene (pLV-PLXNB2: #86237 and pLV-PLXNB2-dECTO).

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Microscopy, Membrane, Labeling, Modification, Fluorescence, Live Cell Imaging, Staining

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Dextran uptake assay of SD2 GSCs labeled with SPY-Actin. Enlarged images of boxed areas are shown below. Box plots show areas of dextran + puncta per cell, with 25–75% quartiles, median (line), and mean (plus sign). n = 85 cells for Ctrl, n = 44 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two-sided. b Top, live cell confocal plane images of SD2 GSCs with side views of z-stacks show intracellular localization of diffuse dextran-Alexa 488 in PB2 KO cells, whereas Ctrl cells contained only dextran + endocytic vesicles. Bottom, histograms of fluorescence profiles show bimodal distribution of dextran-Alexa 488 fluorescence intensities in PB2 KO GSCs (blue and brown arrows). n = 177 cells for Ctrl, n = 161 cells for PB2 KO. Mann–Whitney–Wilcoxon test, two-sided. c , d Left, schematic of myr-palm-GFP or -CFP attached to inner membrane leaflet. Right, live cell images at 72 hr after transfection show fluorescent probes on endomembranes (arrow) in control GSCs, but membrane retention of the probes (arrowhead) in PB2 KO GSCs. For myr-palm-GFP: n = 21 cell membranes for WT; n = 13 cell membranes for PB2 KO. For myr-palm-CFP: n = 6 cell membranes for WT; n = 5 cell membranes for PB2 KO. Two-sided unpaired t-test. Bar graphs show means ± SEM. e Left, schematic of TauSTED super-resolution microscopy of GSCs labeled with MemGlow and NucSpot. Middle, TauSTED live-cell images show reduced endocytic vesicles (arrowheads) in PB2 KO cells compared to Ctrl. Right, box plots show areas of MemGlow + clusters per cell. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 26 cells for Ctrl, n = 13 cells for PB2 KO. Two-sided unpaired t-test. f Working model of regulation of cortical and membrane tension by Plexin-B2, affecting endocytosis in GSCs. Source data are provided as a Source Data file.

Article Snippet: The plasmids for lenti- PLXNB2 expression have been deposited at Addgene (pLV-PLXNB2: #86237 and pLV-PLXNB2-dECTO).

Techniques: Labeling, MANN-WHITNEY, Fluorescence, Membrane, Transfection, Control, Super-Resolution Microscopy

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Illustration of voltage sensitive membrane dye FluoVolt, with fluorescence quenched by electron transfer from electron-rich donor (high inner membrane surface charge), mediated by “molecular wire” in plasma membrane (figure modified after ref. ). b Left, live-cell images show reduced FluoVolt fluorescence in plasma membrane of PB2 KO cells (higher negative charges of inner membrane). Right, box plots of membrane FluoVolt intensity. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 25 cells for Ctrl, n = 27 cells for PB2 KO. Two-sided unpaired t-test. Data represent mean ± SEM. c Top, still images from live-cell imaging show higher FluoVolt fluorescence at rear zone (arrowhead) in Ctrl GSCs when traversing tunnels, more so in 3 than 8 µm tunnel, but not in PB2 KO cells. Calcium chelator BAPTA-AM disrupted polarized FluoVolt pattern. White arrow denotes migration direction. Bottom, bar graphs show the ratio of FluoVolt intensity at rear vs. front during confined migration. n = 15 cells per group for Ctrl. vs KO, one-way ANOVA followed by Tukey’s multiple comparison test. n = 21 cells for vehicle and n = 16 cells for BAPTA-AM, two-sided unpaired t-test. Bar graphs represent mean ± SEM. d Live-cell images and quantifications show the effects of constitutive active (CA) RAP1B-V12 or dominant-negative (DN) RAP1B-N17 on FluoVolt intensity in Ctrl or PB2 KO GSCs. Box plots show 25–75% percentiles, minimal and maximal values (whiskers), median (line), and mean (cross). n = 25 cells per group. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. e Model of Plexin-B2 signaling affecting membrane surface charge and electric field during polarized confined migration, with PIP2 enrichment at cell front and Ca 2+ at rear zone, and asymmetry of FluoVolt and R( +8)-pre-GFP. Source data are provided as a Source Data file.

Article Snippet: The plasmids for lenti- PLXNB2 expression have been deposited at Addgene (pLV-PLXNB2: #86237 and pLV-PLXNB2-dECTO).

Techniques: Membrane, Fluorescence, Clinical Proteomics, Modification, Live Cell Imaging, Migration, Comparison, Dominant Negative Mutation

Journal: Nature Communications

Article Title: Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

doi: 10.1038/s41467-024-55056-6

Figure Lengend Snippet: a Structure model of the extracellular domain of human Plexin-B2 show the locations of lock1 and lock2 mutations predicted to form disulfide bridges that lock the ring structure. b Western blots show absence of mature Plexin-B2 (170 kDa) in PB2 KO GSC, and expression of lock mutants in PB2 KO for both SD2 and SD3. β-actin serves as a loading control. c Still images from videography show passage of GSCs (nuclei visualized by NucSpot) through microchannels by wildtype PB2 rescue construct, but not PB2 lock mutants, nor PB2 with deletion of extracellular domain (dECTO). Chevrons point to 3 µm constrictions. d Box plots show velocity through constrictions (Rescue, n = 20 cells; dECTO, n = 20 cells; Lock1, n = 21 cells; Lock2, n = 17 cells), stalling time at constrictions (Rescue, n = 20 cells; dECTO, n = 14 cells; Lock1, n = 28 cells; Lock2, n = 22 cells), and sum of forward and backward movements constrictions (Rescue, n = 20 cells; dECTO, n = 20 cells; Lock1, n = 21 cells; Lock2, n = 17 cells), with 25–75% quartiles, minimal and maximal values (whiskers), median (line), and mean (cross). One-way ANOVA followed by Dunnett’s multiple comparisons test. e Still images from videography show wildtype but not PB2 mutants restored localization of SPY-actin (arrowhead) at cell rear and MemGlow + endocytic vesicles (arrow) at cell front in GSC traversing 3 µm constrictions (chevrons). f Bar graphs show fluorescence intensity ratio of SPY-actin and MemGlow at rear vs. front of GSCs during confined migration. For SPY-actin: n = 11 cells for Recue; n = 15 cells for dECTO; n = 10 cells for Lock1; n = 10 cells for Lock2. For MemGlow: n = 15 cells for Recue; n = 18 cells for dECTO; n = 10 cells for Lock1; n = 10 cells for Lock2. Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Data represent mean ± SEM. g Model of Plexin-B2 signaling in regulating mechano-electrical coupling of membrane tension and membrane surface charge during polarized confined migration. Note regionalized enrichment of endocytosis/PIP2 at cell front and F-actin/Ca 2+ at rear zone and asymmetry of FluoVolt and R( + 8)-pre-GFP membrane probes. Source data are provided as a Source Data file.

Article Snippet: The plasmids for lenti- PLXNB2 expression have been deposited at Addgene (pLV-PLXNB2: #86237 and pLV-PLXNB2-dECTO).

Techniques: Western Blot, Expressing, Control, Construct, Fluorescence, Migration, Membrane