Research ArticleCytoskeleton

Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters

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Science Signaling  01 May 2018:
Vol. 11, Issue 528, eaam9095
DOI: 10.1126/scisignal.aam9095
  • Fig. 1 GML reduces T cell adhesion, disrupts actin ring formation, and induces filopodia formation at the plasma membrane.

    (A) Activated T cells were treated with or without ethanol (EtOH) or the indicated doses of GML, stained with membrane dye, and incubated on anti-CD3–coated plates. Adhesive cells were imaged and quantified using LI-COR Odyssey software. Top: Images are representative of four independent experiments. Bottom: Normalized fluorescence intensity data are means ± SEM. *P < 0.05 and ***P < 0.001 by one-way analysis of variance (ANOVA) with multiple comparisons. (B) Epifluorescence microscopy of activated T cells treated with ethanol or GML that were stimulated with anti-CD3 and stained with TMR-conjugated phalloidin to visualize actin ring formation. Top: Images are representative of three independent experiments. Bottom: Pixel intensity data along the long medial axis are means ± SEM of 15 cells. Peaks at 4 and 15 μm on the cell axis represent pixel intensity derived from the actin ring. Scale bars, 4 μm. (C) TIRF microscopy of activated T cells restimulated in the presence of either ethanol or GML and stained with TMR-conjugated phalloidin. Images are representative of three independent experiments. Scale bars, 4 μm. (D) Quantification of the number and length of filopodia per cell in (C). Data are means ± SEM of 60 cells. ***P < 0.001 by unpaired t test with Welch’s correction.

  • Fig. 2 GML does not affect the phosphorylation of upstream regulators of actin polymerization.

    (A) Western blotting analysis of phosphorylated Lck and Fyn, FAK, Pyk2, and paxillin was performed at the indicated times after restimulation of activated T cells treated with GML or ethanol. Blots are representative of four independent experiments. (B) Quantification of the Western blots represented in (A). Normalized band intensity data are means ± SEM.

  • Fig. 3 GML-induced filopodia form due to Arp2/3 complex dysfunction and not microtubule or formin inhibition.

    (A to C) TIRF microscopy of activated T cells that were restimulated with anti-CD3 in the presence of ethanol or GML, with or without the indicated small-molecule inhibitors, and stained with TMR-conjugated phalloidin. (A) To disrupt microtubules, cells were treated with 100 μM colchicine. (B) To disrupt formin activity, cells were treated with 10 μM SMIFH2. (C) To disrupt the Arp2/3 complex, cells were treated with 10 μM CK-666. Top: Images are representative of three independent experiments. Bottom: The number and length of filopodia per cell are means ± SEM of 60 cells. Scale bars, 4 μm. **P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparison test. n.s., not significant.

  • Fig. 4 GML does not affect regulatory molecules involved in Arp2/3 complex activation.

    (A) Western blotting analysis of active GTP-bound Rac1 and CDC42 was performed at the indicated times after restimulation of activated T cells in the presence of ethanol or GML. Top: Blots are representative of three independent experiments. Bottom: Normalized band intensity data are means ± SEM. (B and C) Western blotting analysis of total WASp, Arp2, ARPC3, and phosphorylated WASp Y290 was performed at the indicated times after restimulation of activated T cells treated with GML or ethanol. Blots (B) are representative of three independent experiments. Normalized band intensity data (C) are means ± SEM.

  • Fig. 5 GML does not affect the clustering of Arp2, WASp, SLP-76, or ARPC3 at the plasma membrane.

    (A to D) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for (A) Arp2, (B) pWASp, (C) ARPC3, or (D) pSLP-76. Left: Images are representative of three independent experiments. Right: Pixel intensity data are means ± SEM of 60 cells. Scale bars, 4 μm.

  • Fig. 6 GML causes altered localization of WASp, SLP-76, and ARPC3 microclusters and decreased colocalization of LAT and SLP-76 microclusters.

    (A) Co-immunoprecipitation analysis of WASp and ARPC3 was performed at the indicated times after stimulation of activated T cells in the presence of ethanol or GML. Top: Western blots are representative of four independent experiments. Bottom: Normalized band intensity data are means ± SEM. (B) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for Arp2, pWASp, pSLP-76, and ARPC3. Top: Images are representative of GML-treated T cells from three independent experiments. White dashed lines outline the cell periphery. Bottom: Number of fluorescent filopodia. Data are means ± SEM of 60 cells. ***P < 0.001 by one-way ANOVA with Tukey’s multiple comparison test. Scale bars, 2 μm. (C) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for phosphorylated LAT and phosphorylated SLP-76. Top: Images are representative of two independent experiments. Bottom: Pixel intensity and colocalization data are means ± SEM of 60 cells. Scale bars, 4 μm. ***P < 0.001 by unpaired t test with Welch’s correction.

  • Fig. 7 GML does not alter SLP-76 microcluster association with WASp or WAVE2.

    (A) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for phosphorylated WASp and phosphorylated SLP-76. Top: Images are representative of two donors. Bottom: Colocalization data are means ± SEM of 60 cells. Scale bars, 4 μm. (B) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for phosphorylated WAVE and phosphorylated SLP-76. Top: Images are representative of two independent experiments. Scale bars, 4 μm. Bottom: Colocalization data are means ± SEM of 30 cells. (C) TIRF microscopy of activated T cells that were restimulated in the presence of ethanol or GML and stained for phosphorylated WAVE. Images are representative of two independent experiments. Bottom: Number of filopodia. Data are means ± SEM of 30 cells. ****P < 0.0001 by unpaired t test with Welch’s correction.

Supplementary Materials

  • Supplementary Materials for:

    Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters

    Michael S. Zhang, Phuong M. Tran, Alexander J. Wolff, Mikaela M. Tremblay, Micaela G. Fosdick, Jon C. D. Houtman*

    *Corresponding author. Email: jon-houtman{at}uiowa.edu

    This PDF file includes:

    • Fig. S1. Time course of GML-induced filopodia formation.
    • Fig. S2. Model depicting the effects of GML on T cell adhesion.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

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