Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001 . Each point represents average levels of indicated protein at synapse in a single cell. ( B ) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in ( B ) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity. DOI: http://dx.doi.org/10.7554/eLife.04953.007

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Staining, Microscopy, Quantitation Assay, Fluorescence

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Human CD4 T cells were incubated with culture media containing lentiviral particles carrying WASP shRNA or non-specific (control) shRNA for 48 hr ( A ) T cells transduced with WASP shRNA or control shRNA carrying lentiviral particles were incubated with endothelial monolayer for 10 min, fixed and processed for Alexa594-phalloidin (pseudo-colored green) and phospho-HS1 (pseudo-colored red) immuno-staining. The conjugates were then imaged using an EMCCD-coupled spinning disc confocal microscope. Each image represents a single confocal plane of T cell synapse, where the planar endothelial interface is in focus. The area outlined in ‘merge’ panels was further scaled and magnified to show the details with more clarity (bottom panels). The top panels show the image of the field of view in DIC (left image) or fluorescence settings. ( B ) A reduction in WASP levels results in defective phospho-HS1 accumulation at T cell-endothelial cell synapse. The upper graph shows quantitation of phalloidin intensity in the synaptic plane, while the lower graph shows phospho-HS1 levels in the same plane. For both the upper and lower graphs, n1 = 68, n2 = 29, p1 = 0.071, p2 < 0.0001 . This experiment was repeated twice with similar results. ( C ) Model of temporal sequence of events leading to F-actin foci formation and PLCγ signaling at the immunological synapse. Multiple pathways can result in actin polymerization and remodeling at the synaptic interface, contributing to F-actin organization in different SMAC zones. One such pathway involves WAVE2 recruitment by activated LFA1, followed by WAVE2 dependent Arp2/3 complex activation resulting in thick lamellipodial (dSMAC) and lamellar (pSMAC) F-actin meshworks. WAVE2-dependent F-actin pool is required for calcium-dependent calcium entry via the CRAC channel. Additional pathways including MyosinII-mediated actin remodeling is required for maintaining lamellar actin flow and directional persistence of microclusters (MCs) towards the cSMAC, and formin-mediated nucleation of F-actin promotes MTOC docking and stability of synapse. Another pool of F-actin or ‘F-actin foci’ is generated by the activity of WASP protein in the p- and dSMAC zones. Following TCR triggering, WASP is recruited at TCR signalosome via several possible mechanisms – such as via Vav, via NCK, via Zap70 and CrkL mediated WIP release and other effector mechanisms, and, through Fyn or PIP2 or PTP-PEST-binding at the plasma membrane (PM). Once activated, WASP recruits Arp2/3 complex to the MC, which then leads to actin branch nucleation and polymerization at the MC, over and above the local background actin. This process continues even during MC movement in the lamellar region, with a high F-actin turnover at the foci until its delivery to the cSMAC. In the foci, HS1 is recruited via binding both the Arp2/3 complex as well as F-actin, and is subsequently phosphorylated. As a consequence of early TCR signaling, PLCγ1 is also recruited to the MC signalosome, where it is stabilized via interactions with both F-actin, and foci residing HS1. F-actin foci dynamics in the proximity of the plasma membrane further support PLCγ1 phosphorylation, potentially by facilitating its interaction with PM-bound, upstream activators such as Itk. Phosphorylation of PLCγ1 by Itk then triggers phosphoinositide signaling, which in turn initiates calcium ion flux and NFAT1 activation. WASP deficiency or failure to activate Arp2/3 complex by WASPΔC mutant leads to selective loss of nucleation of foci at the MC. As a result, early signaling is not affected, however, both HS1 and PLCγ1 levels are severely reduced at the microcluster sites. The remaining PLCγ1 at synapse allows cell spreading and synapse formation, however, it is not sufficient to achieve calcium flux comparable to the control cells. Direct pharmacological inhibition of Arp2/3 complex using CK666 yields similar results; early TCR signaling is preserved while PLCγ1 phosphorylation and late signaling are severely perturbed. As actin polymerizing processes other than WASP also utilize Arp2/3 Complex, CK666-treated cells show a general reduction in lamellipodial and lamellar actin as well. However, the remaining F-actin levels are sufficient to support early TCR signaling. In contrast, total F-actin depolymerization at the synapse using CytochalasinD results in defects in early as well as late signaling, as has been reported in earlier studies. The image on the bottom shows a maximum intensity projection of synaptic contact interface of a human primary CD4 T cell, acquired using spinning disc confocal microscope. This cell was activated on a bilayer reconstituted with Alexa568 tagged anti-CD3 (red) and ICAM1 (unlabeled), for 2 min, fixed and stained for F-actin (green), and imaged. DOI: http://dx.doi.org/10.7554/eLife.04953.031

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, shRNA, Control, Transduction, Immunostaining, Microscopy, Fluorescence, Quantitation Assay, Sequencing, Activation Assay, Generated, Activity Assay, Binding Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Mutagenesis, Inhibition, Staining

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Example B16-F1 cell shown indicating definition of length and width used in (B; S17A-D): First frame of a movie of LifeAct-EGFP expressing B16-F1 cells to show how length, width and area was quantified: red line indicates area of the lamellipodium. The length was quantified using the Fiji plugin “measure_ROI” ( http://www.optinav.info/Measure-Roi.htm:Measure_Roi_Curve.java ) which measures the length of curved objects (see materials and methods for details). For the width, the line tool in Fiji was used to draw lines (blue lines in (A)) at 90 degree angle to the leading edge to measure width of lamellipodium at 10 roughly equal distance points along the leading edge and the mean of these measurements was used. Scale bar: 20 μm. (B) Quantification of the ratio lamellipodia length/total cell area, One-way ANOVA, Dunnett’s multiple comparisons test; ****, p< 0.0001; **, p=0.0059; F(2,42)=10.84 (C) Quantification of total cellular F-actin (Alexa568 phalloidin) intensity/area ratio in wild type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin. Results are mean ± SEM (error bars) from four independent biological experiments. One-way ANOVA: p=0.0009; Kruskal Wallis statistics 16.44; ***, p=0.0003; **, p=0.0083; ns, p=0.1518; Outliers were removed using the ROUT method (Q=1%) (n analysed/ n outliers = n o removed); WT control: n=67 cells/ n o =0; NHSL1 CRISPR2: n=71 cells/ n o =3; NHSL1 CRISPR2+Myc-NHSL1 SW Mut Rescue: n=76 cells/ n o =5; NHSL1 CRISPR2+Rescue Myc-NHSL1 WT: n=82 cells/ n o =4. (D) Quantification of relative lamellipodial Arp2/3 intensity: Arp2/3 intensity in the lamellipodium was normalized cell-by-cell against Arp2/3 intensity of the whole cell. Kruskal-Wallis test (p=0.0514) and Dunn’s multiple comparisons test: * p=0.0353; ns p>0.9999; in wild-type (n=40 cells, control), NHSL1 CRISPR 2 (n=38 cells) and NHSL1 CRISPR 21 (n=33 cells) B16-F1 cells plated on laminin and stained with anti-ARPC2 (subunit of Arp2/3 complex) antibodies; Results are mean ± SEM (error bars), three independent biological repeats. (E) Quantification of lamellipodial F-actin (Alexa488 phalloidin) intensity/area ratio in B16-F1 wild-type control cells or NHSL1 CRISPR 2 and NHSL1 CRISPR 21 B16-F1 cells plated on laminin. Results are mean ± SEM (error bars), five independent biological repeats, n= control (61), CRISPR 2 (61), CRISPR 21 (43) cells; One-way ANOVA: p=0.0127; F(2,162)=4.486; and Dunnett’s multiple comparisons test: **, p=0.0099. (F) Quantification of lamellipodial F-actin (LifeAct-EGFP) intensity/area ratio in B16-F1 cells transfected with a tri-cistronic plasmid to express Myc-tagged NHSL1 (wild type or Scar/WAVE binding mutant), puromycin resistance and LifeAct-EGFP or empty plasmid (Myc-IRES-Puro-T2A-LifeAct-EGFP) after selection with puromycin plated on laminin. Results are mean ± SEM (error bars), four independent experiments, n= control (14), WT (12), NHSL1-SW mut (13) cells. One-way ANOVA: p=0.0074; F(2,36)=5.636; and Dunnett’s multiple comparisons test: **, p=0.0036; ns p=0.1810. Source data are provided as a Source Data file.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Expressing, CRISPR, Mutagenesis, Binding Assay, Staining, Transfection, Plasmid Preparation, Selection

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: OT1 CTLs expressing shRNA against PTEN (shPTEN) or a control nontargeting shRNA (shNT) were applied to fluorescent micropillars bearing H2-K b -OVA and ICAM1, fixed, and stained with phalloidin (to visualize F-actin). (A) Immunoblot analysis of PTEN expression in shNT and shPTEN CTLs. Actin served as a loading control. (B) Representative images of shNT and shPTEN CTLs, with z-projection images (top views) shown above and corresponding sagittal views below. Cyan dotted lines denote the slicing planes used for the sagittal images. Scale bars = 2 μm. (C) Quantification of sum F-actin intensity on micropillar arrays. Error bars denote SEM. N = 19 for each cell type. P calculated from two-tailed Student’s T-test.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: Expressing, shRNA, Control, Staining, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) The Scar/WAVE complex co-immunoprecipitates with NHSL1. HEK cells were transfected with EGFP-NHSL1, and Myc-Pir121, -Nap-1, -WAVE2, -Abi2. NHSL-1 was immunoprecipitated (pAb 4457) from lysates and co-immunoprecipitation tested on a western blot with Myc and EGFP antibodies. Representative blots from three independent experiments (see Fig. S9A,B for full western blots). (B) and (C) Western blot showing endogenous NHSL1 pulled down with polyclonal NHSL1 (4457) antibody followed by western blotting with (B) Abi1 and (C) Scar/WAVE2 antibodies detecting endogenous co-immunoprecipitation of these proteins in MCF10A (Abi1) and B16-F1 (Scar/WAVE2) cell lysates. Immunoprecipitation using non-immune rabbit IgG served as a negative control. Representative blots from three independent experiments. (D) GST-pull downs using purified Glutathione-sepharose coupled GST-fusion proteins of Abi1 and c-Abl SH3 domains or GST alone from HEK cell lysates that were transfected with EGFP-NHSL1. Following GST-pulldown EGFP-NHSL1 was detected in a western blot with anti-EGFP antibodies. Ponceau staining of membrane reveals GST or GST-tagged Abi- or Abl-SH3 domains used. Representative blots from three independent experiments. (E) Western blot showing immunoprecipitation using NHSL1 polyclonal (4457) antibody or non-immune rabbit IgG control from HEK cell lysates expressing Myc-NHSL1 and all Myc-tagged components of the Scar/WAVE complex, including Myc-Abi1-full-length (E, left), Myc-Abi1-delta-SH3 (E, right). The western blot shows co-immunoprecipitation between NHSL1 and all components of the Scar/WAVE complex only when the Abi SH3 domain is present (Myc-HSPC300 is not shown). Representative blots from three independent experiments. (F) Quantification of band intensity from chemiluminescence from (E) imaged with a CCD camera. Co-immunoprecipitation is reduced by >80%. Bars indicate mean ± SEM (error bars); n = 3; One-way ANOVA: p=0.0002; F(7,16)=8.855; Sidak’s multiple comparisons test: PIR121 **, p=0.0093; Nap1 **, p=0.0028; WAVE2 **, p=0.0074; Abi1 **, p=0.0030. (G) HEK cells were transfected with EGFP-tagged NHSL1 or NHSL1-SH3 binding mutants or EGFP only as negative control and Myc-Abi1. After GFP-trap pulldown of wild type (WT) NHSL1 or different NHSL1-SH3 binding mutants co-precipitation was detected in a western blot with Myc antibody. Representative blots from five independent experiments. Source data are provided as a Source Data file.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Purification, Staining, Expressing, Binding Assay