fcho1 mouse pmcherry c1 cmv n ter mcherry addgene  (Addgene inc)

Journal: Science advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Fig. 4. CIP4 controls actin polymerization around 100-nm plasma membrane deformations. HeLa cells grown on 100-nm nanostructures and treated with siRNAs [(A) to (C) and (G) to (I)] and/or transfected with constructs [(D), (E), and (G) to (I)]. Quantifications of actin [(B), (E), and (G)] or mCherry (H) fluorescence around 100-nm deformations and corresponding representative Airyscan images [(A), (D), and (I)]. (A and B) Effect of CDC42, CIP4, TOCAs (CIP4, FBP17, and FNBP1L), Rac1, clathrin heavy chain, or μ2-adaptin depletion with siRNAs on actin enrichment around deformations. Regions marked by dashed squares, expanded below with individual channels displayed (A, bottom). (C) Immunoblots of CDC42, CIP4, FBP17, FNBP1L, Rac1, clathrin heavy chain, and μ2-adaptin document siRNA knockdown efficiency (uncropped blots, see fig. S13). (D and E) Effect of transient expression of GFP or individual TOCA proteins (mCherry-CIP4, mCherry-FBP17, or GFP-FNBP1L) on actin enrichment around deformations. (F) CIP4 truncation mutants (mCherry-tagged). FL, full-length protein; F-BAR, BAR domain only; F-BAR+, BAR domain with basic region; ∆SH3, lacking SH3 domain; ∆REM1, lacking basic region and REM1 domain; ∆Basic, lacking basic region. (G to I) Rescue experiment. Transient expression of free mCherry, mCherry-tagged full-length CIP4, or truncation mutants in cells where endogenous TOCA proteins are depleted. Number of independent experiments, three [(A), (B), and (G) to (I)] or four [(D) and (E)]. Number of deformations (n) depicted on each graph. Data are means ± SEM; ****P < 0.0001; ***P < 0.001; **P < 0.01 [one-way ANOVAwith Dunnett’s [(B), (E), and (G)] or Tukey’s (H) multiple comparison test]. White arrowheads, colocalization [(A), (D), and (I)]. Scale bars, 10 μm (A) and 2 μm [(D) and (I)].

Article Snippet: CIP4 ΔBasic Mouse pmCherry-C1 CMV N-ter mCherry H. T. McMahon (Cambridge, UK) a.a. 1–308 + 333–547 FBP17 Human pmCherry-C1 CMV N-ter mCherry Addgene #27688 FCHo1 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27690 FCHo2 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27686 FCHSD1 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) FCHSD2 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only Fes Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only FNBP1L Human pEGFP-C1 CMV N-ter GFP P. De Camilli (New Haven, USA) GAS7 Human pEGFP-N1 CMV C-ter GFP C. C.K.

Techniques: Clinical Proteomics, Membrane, Transfection, Construct, Fluorescence, Western Blot, Knockdown, Expressing, Comparison

Journal: Science advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Fig. 5. Local CIP4- and CDC42-dependent actin polymerization is dynamic and promoted by plasma membrane curvature. (A to D) Representative Airyscan time lapses and corresponding quantifications of enrichment ratios of the following pairs around 100-nm deformations over time: (A) GFP-CDC42 and LifeAct-mCherry, (B) GFP-CDC42 and mCherry-CIP4, (C) LifeAct-GFP and mCherry-CIP4, and (D) LifeAct-GFP and PalMyr-mCherry (PalMyr). Thirty-minute time-lapses with 15-s intervals between frames. Region size, 0.85 μm. (E) Correlation coefficients between the pairs of proteins shown in (A) to (D). Number of deformations: PalMyr/LifeAct, n = 65; CIP4/CDC42, n = 118; CIP4/LifeAct, n = 83; LifeAct/CDC42, n = 145. Three independent experiments. (F and G) Monitoring of CDC42-dependent actin polymerization upon deformation of the plasma membrane by FluidFM. (F) Quantification of normalized LifeAct-mCherry fluorescence intensity around FluidFM tip over time upon coex- pression of GFP (control, black), GFP-CDC42-T17N (red), GFP-CDC42-WT (blue), or GFP-CDC42-Q61L (green). Ten-minute time-lapses with 9-s intervals between frames. Number of cells: GFP, n = 20; GFP-CDC42-T17N, n = 18; GFP-CDC42-WT, n = 15; GFP-CDC42-Q61L, n = 17. Two independent experiments. (G) Representative image of FluidFM/confocal experiments upon coexpression of GFP-CDC42-Q61L and LifeAct-mCherry. Region marked by a dashed square surrounding the bead, expanded below at the indicated time points; t = 0 min, initial deformation of the cell membrane. TL, transmitted light (FluidFM cantilever) (representative images of GFP, GFP-CDC42- T17N, and GFP-CDC42-WT in fig. S11, F to H). Data are means ± SEM; ****P < 0.0001 (E, one-way ANOVA with Dunnett’s multiple comparison test). Scale bar, 10 μm (G).

Article Snippet: CIP4 ΔBasic Mouse pmCherry-C1 CMV N-ter mCherry H. T. McMahon (Cambridge, UK) a.a. 1–308 + 333–547 FBP17 Human pmCherry-C1 CMV N-ter mCherry Addgene #27688 FCHo1 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27690 FCHo2 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27686 FCHSD1 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) FCHSD2 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only Fes Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only FNBP1L Human pEGFP-C1 CMV N-ter GFP P. De Camilli (New Haven, USA) GAS7 Human pEGFP-N1 CMV C-ter GFP C. C.K.

Techniques: Clinical Proteomics, Membrane, Fluorescence, Control, Comparison

Journal: Science advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Fig. 4. CIP4 controls actin polymerization around 100-nm plasma membrane deformations. HeLa cells grown on 100-nm nanostructures and treated with siRNAs [(A) to (C) and (G) to (I)] and/or transfected with constructs [(D), (E), and (G) to (I)]. Quantifications of actin [(B), (E), and (G)] or mCherry (H) fluorescence around 100-nm deformations and corresponding representative Airyscan images [(A), (D), and (I)]. (A and B) Effect of CDC42, CIP4, TOCAs (CIP4, FBP17, and FNBP1L), Rac1, clathrin heavy chain, or μ2-adaptin depletion with siRNAs on actin enrichment around deformations. Regions marked by dashed squares, expanded below with individual channels displayed (A, bottom). (C) Immunoblots of CDC42, CIP4, FBP17, FNBP1L, Rac1, clathrin heavy chain, and μ2-adaptin document siRNA knockdown efficiency (uncropped blots, see fig. S13). (D and E) Effect of transient expression of GFP or individual TOCA proteins (mCherry-CIP4, mCherry-FBP17, or GFP-FNBP1L) on actin enrichment around deformations. (F) CIP4 truncation mutants (mCherry-tagged). FL, full-length protein; F-BAR, BAR domain only; F-BAR+, BAR domain with basic region; ∆SH3, lacking SH3 domain; ∆REM1, lacking basic region and REM1 domain; ∆Basic, lacking basic region. (G to I) Rescue experiment. Transient expression of free mCherry, mCherry-tagged full-length CIP4, or truncation mutants in cells where endogenous TOCA proteins are depleted. Number of independent experiments, three [(A), (B), and (G) to (I)] or four [(D) and (E)]. Number of deformations (n) depicted on each graph. Data are means ± SEM; ****P < 0.0001; ***P < 0.001; **P < 0.01 [one-way ANOVAwith Dunnett’s [(B), (E), and (G)] or Tukey’s (H) multiple comparison test]. White arrowheads, colocalization [(A), (D), and (I)]. Scale bars, 10 μm (A) and 2 μm [(D) and (I)].

Article Snippet: CIP4 ΔBasic Mouse pmCherry-C1 CMV N-ter mCherry H. T. McMahon (Cambridge, UK) a.a. 1–308 + 333–547 FBP17 Human pmCherry-C1 CMV N-ter mCherry Addgene #27688 FCHo1 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27690 FCHo2 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27686 FCHSD1 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) FCHSD2 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only Fes Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only FNBP1L Human pEGFP-C1 CMV N-ter GFP P. De Camilli (New Haven, USA) GAS7 Human pEGFP-N1 CMV C-ter GFP C. C.K.

Techniques: Clinical Proteomics, Membrane, Transfection, Construct, Fluorescence, Western Blot, Knockdown, Expressing, Comparison

Journal: Science advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Fig. 5. Local CIP4- and CDC42-dependent actin polymerization is dynamic and promoted by plasma membrane curvature. (A to D) Representative Airyscan time lapses and corresponding quantifications of enrichment ratios of the following pairs around 100-nm deformations over time: (A) GFP-CDC42 and LifeAct-mCherry, (B) GFP-CDC42 and mCherry-CIP4, (C) LifeAct-GFP and mCherry-CIP4, and (D) LifeAct-GFP and PalMyr-mCherry (PalMyr). Thirty-minute time-lapses with 15-s intervals between frames. Region size, 0.85 μm. (E) Correlation coefficients between the pairs of proteins shown in (A) to (D). Number of deformations: PalMyr/LifeAct, n = 65; CIP4/CDC42, n = 118; CIP4/LifeAct, n = 83; LifeAct/CDC42, n = 145. Three independent experiments. (F and G) Monitoring of CDC42-dependent actin polymerization upon deformation of the plasma membrane by FluidFM. (F) Quantification of normalized LifeAct-mCherry fluorescence intensity around FluidFM tip over time upon coex- pression of GFP (control, black), GFP-CDC42-T17N (red), GFP-CDC42-WT (blue), or GFP-CDC42-Q61L (green). Ten-minute time-lapses with 9-s intervals between frames. Number of cells: GFP, n = 20; GFP-CDC42-T17N, n = 18; GFP-CDC42-WT, n = 15; GFP-CDC42-Q61L, n = 17. Two independent experiments. (G) Representative image of FluidFM/confocal experiments upon coexpression of GFP-CDC42-Q61L and LifeAct-mCherry. Region marked by a dashed square surrounding the bead, expanded below at the indicated time points; t = 0 min, initial deformation of the cell membrane. TL, transmitted light (FluidFM cantilever) (representative images of GFP, GFP-CDC42- T17N, and GFP-CDC42-WT in fig. S11, F to H). Data are means ± SEM; ****P < 0.0001 (E, one-way ANOVA with Dunnett’s multiple comparison test). Scale bar, 10 μm (G).

Article Snippet: CIP4 ΔBasic Mouse pmCherry-C1 CMV N-ter mCherry H. T. McMahon (Cambridge, UK) a.a. 1–308 + 333–547 FBP17 Human pmCherry-C1 CMV N-ter mCherry Addgene #27688 FCHo1 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27690 FCHo2 Mouse pmCherry-C1 CMV N-ter mCherry Addgene #27686 FCHSD1 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) FCHSD2 Mouse pEGFP-C2 CMV N-ter GFP Z. Xu (Shandong, China) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) Fer Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only Fes Human pEGFP-C1 CMV N-ter GFP K. Gould (Nashville, USA) BAR domain only FNBP1L Human pEGFP-C1 CMV N-ter GFP P. De Camilli (New Haven, USA) GAS7 Human pEGFP-N1 CMV C-ter GFP C. C.K.

Techniques: Clinical Proteomics, Membrane, Fluorescence, Control, Comparison

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Plasmids containing BAR domain protein sequences used in this study.

Article Snippet: FCHo1 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27690 , .

Techniques:

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: HeLa cells grown on 100-nm nanostructures and treated with siRNAs [(A) to (C) and (G) to (I)] and/or transfected with constructs [(D), (E), and (G) to (I)]. Quantifications of actin [(B), (E), and (G)] or mCherry (H) fluorescence around 100-nm deformations and corresponding representative Airyscan images [(A), (D), and (I)]. ( A and B ) Effect of CDC42, CIP4, TOCAs (CIP4, FBP17, and FNBP1L), Rac1, clathrin heavy chain, or μ2-adaptin depletion with siRNAs on actin enrichment around deformations. Regions marked by dashed squares, expanded below with individual channels displayed (A, bottom). ( C ) Immunoblots of CDC42, CIP4, FBP17, FNBP1L, Rac1, clathrin heavy chain, and μ2-adaptin document siRNA knockdown efficiency (uncropped blots, see fig. S13). ( D and E ) Effect of transient expression of GFP or individual TOCA proteins (mCherry-CIP4, mCherry-FBP17, or GFP-FNBP1L) on actin enrichment around deformations. ( F ) CIP4 truncation mutants (mCherry-tagged). FL, full-length protein; F-BAR, BAR domain only; F-BAR+, BAR domain with basic region; ∆SH3, lacking SH3 domain; ∆REM1, lacking basic region and REM1 domain; ∆Basic, lacking basic region. ( G to I ) Rescue experiment. Transient expression of free mCherry, mCherry-tagged full-length CIP4, or truncation mutants in cells where endogenous TOCA proteins are depleted. Number of independent experiments, three [(A), (B), and (G) to (I)] or four [(D) and (E)]. Number of deformations ( n ) depicted on each graph. Data are means ± SEM; **** P < 0.0001; *** P < 0.001; ** P < 0.01 [one-way ANOVA with Dunnett’s [(B), (E), and (G)] or Tukey’s (H) multiple comparison test]. White arrowheads, colocalization [(A), (D), and (I)]. Scale bars, 10 μm (A) and 2 μm [(D) and (I)].

Article Snippet: FBP17 , Human , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27688 , .

Techniques: Transfection, Construct, Fluorescence, Western Blot, Knockdown, Expressing, Comparison

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: ( A to D ) Representative Airyscan time lapses and corresponding quantifications of enrichment ratios of the following pairs around 100-nm deformations over time: (A) GFP-CDC42 and LifeAct-mCherry, (B) GFP-CDC42 and mCherry-CIP4, (C) LifeAct-GFP and mCherry-CIP4, and (D) LifeAct-GFP and PalMyr-mCherry (PalMyr). Thirty-minute time-lapses with 15-s intervals between frames. Region size, 0.85 μm. ( E ) Correlation coefficients between the pairs of proteins shown in (A) to (D). Number of deformations: PalMyr/LifeAct, n = 65; CIP4/CDC42, n = 118; CIP4/LifeAct, n = 83; LifeAct/CDC42, n = 145. Three independent experiments. ( F and G ) Monitoring of CDC42-dependent actin polymerization upon deformation of the plasma membrane by FluidFM. (F) Quantification of normalized LifeAct-mCherry fluorescence intensity around FluidFM tip over time upon coexpression of GFP (control, black), GFP-CDC42-T17N (red), GFP-CDC42-WT (blue), or GFP-CDC42-Q61L (green). Ten-minute time-lapses with 9-s intervals between frames. Number of cells: GFP, n = 20; GFP-CDC42-T17N, n = 18; GFP-CDC42-WT, n = 15; GFP-CDC42-Q61L, n = 17. Two independent experiments. (G) Representative image of FluidFM/confocal experiments upon coexpression of GFP-CDC42-Q61L and LifeAct-mCherry. Region marked by a dashed square surrounding the bead, expanded below at the indicated time points; t = 0 min, initial deformation of the cell membrane. TL, transmitted light (FluidFM cantilever) (representative images of GFP, GFP-CDC42-T17N, and GFP-CDC42-WT in fig. S11, F to H). Data are means ± SEM; **** P < 0.0001 (E, one-way ANOVA with Dunnett’s multiple comparison test). Scale bar, 10 μm (G).

Article Snippet: FBP17 , Human , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27688 , .

Techniques: Clinical Proteomics, Membrane, Fluorescence, Control, Comparison

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Plasmids containing BAR domain protein sequences used in this study.

Article Snippet: FBP17 , Human , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27688 , .

Techniques:

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Other plasmids used in this study.

Article Snippet: FBP17 , Human , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27688 , .

Techniques:

Journal: Science Advances

Article Title: Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling

doi: 10.1126/sciadv.ade1660

Figure Lengend Snippet: Plasmids containing BAR domain protein sequences used in this study.

Article Snippet: FCHo2 , Mouse , pmCherry-C1 , CMV , N-ter mCherry , Addgene #27686 , .

Techniques: