Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: (A and B) Schematic of the model transmembrane protein FRTM-Ezrin-AFBD (A) and the mutant FRTM-Ezrin-R579A (FRTM-Ez-AFBD*) (B) that impairs Ezrin-AFBD ability to interact with actin . (C–F) Representative intensity and steady-state anisotropy images and scatter dot plots with mean anisotropy of ROIs obtained from CHO cells stably expressing either FRTM-Ez-AFBD or FRTM-Ez-AFBD* as indicated. The cells were labeled with fluorescent folate, Pteroyl-lysyl-Bodipy(PLB) and plated on FN (blue, green) or glass (red, orange) prior to imaging in the absence (C and D) or after pre-treatment (E and F) with either 20 μM PP2 and 10 μM PF-573228 (red) or 10 μM SMIFH2 (green) or with the vehicle (DMSO; blue). (G–I) Schematic (G) of the supported lipid bilayer functionalized with cRGD that was prepared either on plain (continuous SLB; top) or on 5-nm-tall and 100-nm-wide chromium patterned (nanopatterned SLB, bottom) glass surfaces. (H and I) GFP-GPI-expressing CHO cells plated on glass (red) or on FN (blue) or treated with 10 mM mβCD on FN (green) or plated on either continuous SLBs with mobile ligand (magenta) or SLBs assembled on chromium nano-patterned surfaces. ROIs were drawn either on the pattern (orange) where the ligand is transiently immobile or from regions outside (brown) where the ligand is mobile. Scale bar 10μm in all panels. All error bars represent SD. n.s. p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size and p values are provided in . See also .

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Mutagenesis, Stable Transfection, Expressing, Labeling, Imaging

Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: (A–J) Representative intensity and steady-state anisotropy images (A, C, E, G, and I) and scatter dot plot with mean anisotropy values (B, D, F, H, and J) of ROIs obtained from (A and B) vinculin-deficient cells (Vin −/− ) transfected with GFP-GPI (blue, orange) or co-transfected with mCherry-vinculin (+Vin-WT; red, green) and plated on FN or subsequently treated with 10 mM mβCD (orange, green). (C and D) Talin1-deficient cells without (Talin1 −/− ; blue, red) or with co-transfection with Talin2 shRNA (+Talin2 shR; green, orange) and re-plated onto FN after labeling with Alexa-568-FLAER prior to (blue, green) or post-treatment with 10 mM mβCD (red, orange). (E and F) Vin −/− cells alone (blue) or transiently transfected with GFP tagged Vin-WT (green), Vin-A50I (orange), or Vin-A50I-CA (brown) and plated onto FN after labeling with Alexa-568-FLAER. (G and H) Vin −/− cells were transiently transfected with FRTM-Ez-AFBD (FR-EZ; green) or with FR-Ez-AFBD* mutant (FR-EZ*; red), without (open circles) or with Vin-WT (closed circles) and re-plated onto FN after labeling with PLB. (I and J) Vin −/− cells alone (blue) or transfected with Lact C2-Ez-AFBD YFP (red) were labeled with Alexa-568-FLAER and re-plated on FN and directly labeled or treated with 10 mM mβCD (+mβCD; green). Dotted magenta lines in all images outline the transfected cells expressing the indicated constructs. Scale bar, 10 μm in all panels. All error bars represent SD. n.s. p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size and p values are provided in . See also .

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Transfection, Cotransfection, shRNA, Labeling, Mutagenesis, Expressing, Construct

Journal: Cell

Article Title: Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading

doi: 10.1016/j.cell.2019.04.037

Figure Lengend Snippet: Key Resources Table

Article Snippet: Lact C2 Ezrin AFBD-YFP , Protein Technology Core (C-CAMP,Bangalore,India) ( ) , N/A.

Techniques: Purification, Transduction, Recombinant, Clinical Proteomics, Avidin-Biotin Assay, Membrane, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Mutagenesis, Derivative Assay, Control, Construct, shRNA, Software