Journal: bioRxiv

Article Title: Inhibiting ezrin triggers formin-mediated actin remodelling reducing cellular mechano-protection

doi: 10.1101/2025.07.22.666169

Figure Lengend Snippet: A. i. Representative images of fluorescence recovery in CHO cells transiently expressing myosin IIA-mCherry. ii. Comparison of recovery characteristics and half-life of myosin IIa-mCherry upon ezrin inhibition. Scale: 5 µm. N= 3 independent experiments, n Control = 40 cells, n EzrInh = 42 cells. B. i. Representative FLIM images captured at the basal as well as mid-plane of CHO cells stained with Flipper-TR (1 µM, 50 min). Scale: 10 µm. Zoomed-in images show a part of the cell membrane ( top ) along with the corresponding ROI of 2-pixel thickness ( bottom ) used for analysis. Scale: 1 µm. ii. Comparisons of lifetimes at base and mid plane upon ezrin inhibition. iii. Comparison of ratio of lifetimes (L2/L1) on EzrInh treatment. iv. Fluorescence lifetime increases upon hypo-osmotic shock. N= 3 independent experiments, n Control = 28 cells, n EzrInh = 30 cells. v. Ratio of lifetimes at mid plane (L2) and base (L1). N=3 independent experiments, n Control = 57 cells, n EzrInh = 57 cells. iv. C. i. Representative IRM images of CHO cells with corresponding FBR maps of EzrInh CHO cells upon hypo-osmotic shock. Scale: 10 µm. ii. Comparison of fold change in ratio of tension at cell edge versus inside. N=4 independent experiments, n Control = 23 cells, n EzrInh = 24 cells with same cells followed over time. Error bars represent standard errors. D. Representative TIRF images of CHO cells transiently expressing LifeAct mCherry before and after hypo-osmotic shock. Scale: 10 µm. ii. Zoomed-in view showing actin dynamics when hypo-osmotic shock is administered on ezrin-inhibited cells. Scale: 2 µm. iii. Kymograph generated across line ROI (black dotted line in ii) represent evolution of protrusions (white arrows) and corresponding retractions (yellow arrows). X-axis represents distance across ROI (1 pixel= 65 nm) while time on Y-axis (1 pixel= 2 sec). Scale: 2 µm (X-axis), 60 sec (Y-axis). E. i. Comparison of number of protrusions per cell. ii. Quantification of percentage of cells showing high protrusion numbers (>8 protrusions per cell). iii. Comparison of absolute ratio of rate of retraction to rate of protrusion. N= 6 independent experiments, n Control = 15 cells, n EzrInh = 20 cells. Statistical significance was determined by Mann Whitney U-test where *p < 0.05, **p < 0.01, ***p < 0.001 and ns p > 0.05.

Article Snippet: For live cells visualization of F-actin, well spread CHO cells growing on glass cover-slips were transfected with pDEST/Life-Act-mCherry-N1(a gift from Rowin Shaw, Addgene, plasmid#40908).

Techniques: Fluorescence, Expressing, Comparison, Inhibition, Control, Staining, Membrane, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: Inhibiting ezrin triggers formin-mediated actin remodelling reducing cellular mechano-protection

doi: 10.1101/2025.07.22.666169

Figure Lengend Snippet: A. Representative TIRF images of F-actin labelled with Alexa Fluor 568 Phalloidin ROCKInh (10 µM, 1 hr) or SMIFH2 (10 µM, 1 hr) pre-treated cells on EzrInh treatment. B. i-iv. Comparison of total number of stress fibres and spread area on EzrInh treatment. v-viii. Comparison of number of SFPeri and SFCort on ezrin inhibition. ix-xii. Comparison of mean intensities of SFPeri and SFCort. N= 3 independent experiments. n ROCKInh = 157 cells, n ROCKInh+EzrInh = 172 cells, n SMIFH2 = 133 cells, n SMIFH2+EzrInh = 94 cells. Scale: 10 µm. Statistical significance was determined by Mann Whitney U-test where *p < 0.05, **p < 0.01, ***p < 0.001 and ns p > 0.05 with Bonferroni correction. C. Rupture assay of CHO cells upon hypo-osmotic shock on EzrInh treatment in SMIFH2 pre-treated cells. i. Representative epi-fluorescence images of Calcein AM loaded cells before and after hypo-osmotic shock. ii. Comparison of rupture propensity (quadrant-wise), rupture time (all cells) and rupture time and percentage of cells rupturing across three time-scales. N= 3 independent experiments, No. of quadrants: Q SMIFH2 = 20, Q SMIFH2+EzrInh = 24; Total number of cells n1, Total no. of ruptures: nrup SMIFH2 = 171, nrup SMIFH2+EzrInh = 8. D. Representative TIRF images of SMIFH2 pre-treated CHO cells on EzrInh treatment, transiently expressing LifeAct mCherry, before and after hypo-osmotic shock. Scale: 10 µm. ii. Kymograph generated across line ROI (yellow line ROI in ii) representing evolution of protrusions (white arrows) and corresponding retractions (yellow arrows). X-axis represents distance across ROI (1 pixel= 65 nm) while time on Y-axis (1 pixel= 2 sec). Scale: 2 µm (X-axis), 60 sec (Y-axis). iii. Quantification of percentage of cells showing high protrusion numbers (>8 protrusions per cell). Also shown comparison of absolute ratio of rate of retraction and rate of protrusion. Error bars represent standard deviation. N= 2 independent experiments, n SMIFH2 = 5 cells, n SMIFH2+EzrInh = 7 cells. Statistical significance was determined by Mann Whitney U-test where *p < 0.05, **p < 0.01, ***p < 0.001 and ns p > 0.05.

Article Snippet: For live cells visualization of F-actin, well spread CHO cells growing on glass cover-slips were transfected with pDEST/Life-Act-mCherry-N1(a gift from Rowin Shaw, Addgene, plasmid#40908).

Techniques: Comparison, Inhibition, MANN-WHITNEY, Fluorescence, Expressing, Generated, Standard Deviation

Journal: Development (Cambridge, England)

Article Title: Transfection of Capsaspora owczarzaki , a close unicellular relative of animals

doi: 10.1242/dev.162107

Figure Lengend Snippet: Capsaspora expression cassettes and live imaging of transfected cells . (A) Cytosolic marker cassettes expressing mCherry (A′) or Venus (A″) fluorescent proteins. (B) Plasma membrane marker cassette containing the Capsaspora Src2 NMM fused to mCherry (B′). (C) Actin marker cassette containing Lifeact fused to mCherry (C′). (D) Nuclear marker cassette containing Capsaspora histone H2B ( CoH2B ) fused to Venus (D′). EF-1α promoter (arrows) and terminator (grey boxes) and single-cut restriction enzymes are shown. Cells in (A) and (D) were imaged using wide-field fluorescence microscopy. The cell in B was imaged using a Spinning Disk confocal microscope and the cell in C was imaged using a confocal laser scanning microscope. Dashed line indicates the cell body. Scale bars: 5 μm.

Article Snippet: Data availability Capsaspora expression vectors have been deposited in Addgene (www.addgene.org) under the following accession numbers: pONSY (empty) (111873), pONSY-mCherry (111874), pONSY-Venus (111875), pONSY-Lifeact:mCherry (111876), pONSY-CoH2B:Venus (111877) and pONSY-CoNMM:mCherry (111878).

Techniques: Expressing, Imaging, Transfection, Marker, Fluorescence, Microscopy, Laser-Scanning Microscopy

Journal: Development (Cambridge, England)

Article Title: Transfection of Capsaspora owczarzaki , a close unicellular relative of animals

doi: 10.1242/dev.162107

Figure Lengend Snippet: Transfection efficiency analysis of Capsaspora . (A) Flow cytometry distribution of pONSY-Venus transfected cells. Area selected (P+) represents the Venus-positive population. (B) Flow cytometry distribution of pONSY-mCherry transfected cells. Area selected (P+) represents the mCherry-positive population. (C) Percentage of positive cells in single transfection. The box plot represents the transfection efficiency distribution over seven independent experiments with at least six technical replicates each ( n =4.9 M cells). (D) Percentage of positive cells from a paired experiment with six technical replicates, transfecting either pONSY-Venus or pONSY-mCherry. Error bars represent s.d. ( P =0.5625, Wilcoxon Signed Rank Test). (E) Wide-field fluorescence microscopy of a live cell co-transfected with pONSY-Venus and pONSY-mCherry. (F) Flow cytometry distribution of pONSY-Venus and pONSY-mCherry co-transfected cells. Cell population was divided into quartiles: negative cells (Q1), fluorescent cells expressing mCherry only (Q2), co-transfected cells expressing both fluorescent proteins (Q3), and fluorescent cells expressing Venus only (Q4). (G) Relative percentage of positive cells co-transfected with pONSY-Venus and pONSY-mCherry; expressing both fluorescent proteins (double), mCherry only or Venus only, calculated from the total number of positive cells in seven independent experiments with six replicates each ( n =120,000 cells). Scale bar: 5 µm.

Article Snippet: Data availability Capsaspora expression vectors have been deposited in Addgene (www.addgene.org) under the following accession numbers: pONSY (empty) (111873), pONSY-mCherry (111874), pONSY-Venus (111875), pONSY-Lifeact:mCherry (111876), pONSY-CoH2B:Venus (111877) and pONSY-CoNMM:mCherry (111878).

Techniques: Transfection, Flow Cytometry, Fluorescence, Microscopy, Expressing

Journal: Development (Cambridge, England)

Article Title: Transfection of Capsaspora owczarzaki , a close unicellular relative of animals

doi: 10.1242/dev.162107

Figure Lengend Snippet: Live imaging of transfected Capsaspora cells. (A) Wide-field fluorescence microscopy of live cells co-transfected with pONSY-CoNMM:mCherry and pONSY-CoH2B:Venus, and live cells co-transfected with pONSY-Lifeact:mCherry and pONSY-CoH2B:Venus. CoNMM:mCherry labelling is presented as a maximum projection of the cell. Dashed lines indicate cell bodies. (B) Time-points on a Capsaspora cell transfected with pONSY-CoNMM:mCherry imaged using wide-field fluorescence microscopy. Filopodia attached to the substrate are in focus. A retracting filopodia can be observed (dotted line), whereas four filopodia are broken (arrowheads). (C) Maximum-intensity projections in each axis of a cell transfected with pONSY-CoNMM:mCherry. Colour scale represents depth through the projection. Imaging was performed using confocal microscopy. (D) z-stack on a Capsaspora cell transfected with pONSY-Lifeact:mCherry imaged using confocal microscopy. Actin bundles can be observed shaping the cell in a basket-like structure that is hollow in the middle. (E) Full z-stack maximum intensity projection of cell in (D). Cells in (C-E) were imaged using a Spinning Disk confocal microscope. Scale bars: 5 µm.

Article Snippet: Data availability Capsaspora expression vectors have been deposited in Addgene (www.addgene.org) under the following accession numbers: pONSY (empty) (111873), pONSY-mCherry (111874), pONSY-Venus (111875), pONSY-Lifeact:mCherry (111876), pONSY-CoH2B:Venus (111877) and pONSY-CoNMM:mCherry (111878).

Techniques: Imaging, Transfection, Fluorescence, Microscopy, Confocal Microscopy