Journal: bioRxiv

Article Title: Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

doi: 10.1101/2025.03.17.643603

Figure Lengend Snippet: (a) Alignment of the amino acid sequence of caspase-3 IDR, caspase-7 IDR, IDR mutants. Acidic amino acids are shown in red. Basic amino acids are shown in blue. Caspase recognition sites are shown in boxes. Mutated amino acids are highlighted in gray. (b, c) Basic and hydrophobic (BH)-scores of caspase-3 and caspase-7. The red line indicates a threshold of 0.6. Caspase-3 IDR and caspase-7 IDR are masked with orange and green blocks, respectively. (d) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 45 cells), caspase-7 KO (n = 37 cells), with 7IDR-3-FLAG (n = 35 cells), 7IDR ED/A -3-FLAG (n = 17 cells), 7IDR KR/A -3-FLAG (n = 34 cells). (e) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 15 cells), caspase-7 KO (n = 44 cells), with 7IDR-3-FLAG (n = 24 cells), 7IDR D23A -3-FLAG (n = 31 cells). (f) A schematic image illustrating differential interference contrast (DIC), TIRF, and epifluorescence observation (top). Representative images of mScarlet3 expressing HeLa cells (bottom). Scale bar: 50 μm. (g) The ratio of TIRF fluorescence intensity to epifluorescence intensity, normalized to mScarlet3 (mS3) alone. (h) Lipid overlay assays with recombinant Lact-C2-FLAG, 7IDR D23A -3 CtoG -FLAG, 7IDR D23A, ED/A - 3 CtoG -FLAG, 7IDR Δ23 -3 CtoG -FLAG, and 7IDR Δ23, KR/A -3 CtoG -FLAG. (i) A schematic diagram depicting Lact-C2 interacting with phosphatidylserine (PS) in the inner leaflet of the PM (top). Representative images of Lact-C2 fused with HaloTag7 (bottom). Scale bars: 10 μm. (j) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 27 cells), caspase-7 KO (n = 19 cells), with 7IDR-3-FLAG (n = 16 cells), 7IDR-3-FLAG + HaloTag7-Lact-C2 (n = 18 cells), 7IDR-3-FLAG + HaloTag7-Lact-C2 AAA (n = 26 cells). (k) Δt 1/2 of PM Dual FRET transiently expressed in WT (n = 27 cells), with HaloTag7-Lact-C2 (n = 29 cells), HaloTag7-Lact-C2 AAA (n = 25 cells). For all figures, dot plots show the mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s comparison test. NS: P > 0.05; *: P < 0.05; **: P < 0.01; ****: P < 0.0001.

Article Snippet: For pcDNA3.1-HaloTag7-Lact-C2 and pcDNA3.1-HaloTag7-Lact-C2 AAA , the coding sequence of Lact-C2 was PCR-amplified using primers for Lact-C2 or mutation-introducing primers (W26A; TGG to GCC, W33A; TGG to GCC, F34A; TTT to GCC) from Lact-C2-GFP (#22852; Addgene).

Techniques: Sequencing, Expressing, Fluorescence, Recombinant, Comparison

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