Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Representative confocal fluorescence microscopy time-lapse images of HeLa Lifeact-mCherry (Actin, top row) MYH9-eGFP (NMIIA, bottom row) confined to 3μm inside a nonadhesive 2D confiner device. Time stamp, time from confinement in seconds. Square, magnification of a transient bleb protrusion-retraction cycle. Arrow, formation of a stable bleb. Scale bar, 10 μm. B: Left, schematic representation of the morphological features of blebs formed upon confinement. The immediate response of non-adherent cells to confinement is the formation of round, transient blebs, represented in grey. After 15 seconds approximately, elongated stable blebs, represented in orange, start to appear. Centre, individual bleb lifetime (the difference between the time of formation and time when the bleb is completely retracted, Welch’s P <0.001) for round (n = 76) and elongated blebs (n = 80). Right, quartile intervals and mean (cross) of the aspect ratio (length/width) for all blebs in the first few time points (15s) versus all blebs after the first time points. For clarity, the boxplot excludes the outliers. C: Top, representative fluorescence confocal images of a round (Round bleb, grey, left column) and an elongated bleb (Elongated, orange, right column) from 3 μm-confined HeLa MYH9 -eGFP (NMIIA, left image) Lifeact-mCherry (Actin, right image). Scale bar, 5 μm. Bottom, averaged cortical actin (magenta) or NMIIA density (cyan), binned and normalized by position along bleb length, for representative round (n = 10) and elongated (n = 8) blebs. Shaded curves, individual data. D: Representative fluorescence confocal time-lapse images of HeLa ActB -GFP cells confined to 3μm inside a PLL-coated 2D confiner device. Time stamp, time from confinement in seconds. Arrow, stable bleb separating from the main cell body. Scale bar, 10 μm. E: Boxplot of front protrusion and actin flow average speed in stable blebs inside a PLL- g -PEG-coated microfluidic chamber (magenta, “PLL- g -PEG”), or inside a PLL-coated microfluidic chamber (cyan, “PLL”) (box line, median; box cross, mean; n PLL- g -PEG = 19 cells, n PLL = 23 cells; P value, Welch’s t test). F: Representative fluorescence confocal images of cytoplasts originated from HeLa ActB-GFP blebs confined to 3μm inside a PLL-coated (adhesive) microfluidic chamber. Scale bar, 10 μm.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Diagram representing the analysis of panel S2B. The coefficient of variation (CV) serves a proxy of the sharpness or focus of the image of the bleb front. B: Coefficient of variation of the actin signal measured at the bleb front in ActB-GFP live hNA TIRF (also labelled as actin-GFP, cyan, n = 55 blebs), F-tractin-GFP live hNA TIRF (cyan, n = 13 blebs), lifeact-mCherry live hNA TIRF (cyan, n = 35 blebs), and fixed phalloidin Alexa Fluor 488 imaged on a confocal spinning disk (dark grey, n = 41 blebs). P values, Welch’s T test. C: Representative live hNA TIRF image of 3 μm-confined HeLa ActB -GFP cells. Bleb square, zoom of the bleb front. Scale bar, 10 μm. D: Representative live hNA TIRF image of 3 μm-confined HeLa cells transfected with F-tractin-GFP. Scale bar: 10 μm. E: Representative live hNA TIRF image of 3 μm-confined HeLa cells transfected with lifeact-mCherry. Scale bar: 10 μm. F: Representative fixed confocal spinning disk image of 3 μm-confined HeLa cells labelled with Alexa Fuor-488 phalloidin. Scale bar: 10 μm. G: Left, representative live high numerical aperture TIRF image of 3 μm-confined HeLa cells transfected with alpha-actinin-GFP. Black arrows, filament-like pattern. Scale bar, 10μm. Middle, representative live hNA TIRF image of 3 μm-confined HeLa ActB -GFP cells transfected with alpha-actinin-mCherry. Scale bar, 10 μm. Right, zoomed image. Black arrows, sites of filament overlapping and accumulation of alpha-actinin. Scale bar, 1 μm. H: Background-subtracted intensity of actin (cyan) and alpha-actinin (red) scanned along the filament pointed in panel S2G. Right, interpretation of the intensity levels.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Transfection

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Left and middle panels, representative high numerical aperture TIRF images of 3 μm-confined HeLa ActB -GFP cells. Right panels, analysis of the actin network. Top panels, stable bleb protruding at the front. Bottom panels, stable bleb protruding to the left side. Red lines and label “I”, actin filaments not linked to the main actin network. Blue lines and label “II”, main actin network. Scale bar, 10μm. B: Top, representative high numerical aperture TIRF image of a stable bleb from 3 μm-confined HeLa ActB -GFP cells. Scale bar, 10 μm. Bottom, zoom of the regions marked with black squares. Arrows, actin filaments not connected to the main network. Scale bar, 1 μm. C: Left, histogram of the rotational velocity ( v rot , rad/s) of actin filaments not connected to the main network (fragments, red), or connected to the network (network, cyan). Right, representative high numerical aperture TIRF color-coded time projections of actin filaments not connected to the main network, in 3 μm-confined HeLa ActB -GFP cells. Scale bar, 1μm. D: Left, mean square angular displacement (MSAD, rad 2 ) of actin filaments not connected to the main network (fragments, red), or connected to the network (network, cyan). Right, mean square displacement (MSD, μm 2 ) of actin filaments not connected to the main network (fragments, red), or connected to the network (network, cyan). E: Representative high numerical aperture TIRF time-lapse images of a filament elongation event, marked with black arrows. Scale bar, 1μm. F: Left, kymograph of the branching event from panel 2E. Scale bar, 1μm/s. Right, histogram of filament branching elongation speed (μm/s) and branching angle (°). Label, mean value ± standard deviation. G: Representative high numerical aperture TIRF time-lapse images of a filament crosslinking event, marked with black arrows. Scale bar: 1μm. H: Representative high numerical aperture TIRF time-lapse images of a branch growth and collapse event, marked with black arrows. Scale bar: 1μm. I: Representative high numerical aperture TIRF time-lapse images of a bleb with network branching (top) or network seeding (bottom) as main assembly mechanism. J: Frequency of nucleation of network segments by branching (cyan) or seeding (red), measured as number of events per μm 2 min at the bleb front.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Representative hNA TIRF image of 3 μm-confined HeLa ActB -GFP (TIRF actin-GFP, left) cells transfected with mApple-MyosinIIA (TIRF NMIIA-mApple, right). Scale bar, 10 μm. B: Actin (black, mean ± s.d.) and NMIIA (blue, mean ± s.d.) density profile along bleb length, averaged over the width, time, and several blebs (N = 3, n bleb = 20, n t = 2120). C: Left, time-averaged PIV of a single bleb (n bleb = 1, n t = 201). Right, time-averaged PIV divergence field of a single bleb (nbleb = 1, n t = 201). D: PIV horizontal component (v x , black, mean ± s.d.) and PIV divergence (blue, mean ± s.d.) along bleb length, averaged over the width, time, and several blebs (N = 3, n bleb = 20, n t = 2120). E: Left, time-averaged net actin turnover of a single bleb (n bleb = 1, n t = 201). Right, time averaged net NMIIA turnover of a single bleb (n bleb = 1, n t = 201). F: Net actin turnover (black, mean ± s.d.) and net NMIIA turnover (blue, mean ± s.d.) along bleb length, averaged over the width, time, and several blebs (N = 3, n bleb = 20, n t = 2120). G: Left, time-averaged local correlation C(r 0 ) of a single bleb (n bleb = 1, n t = 201). Right, time-averaged correlation length C ( r ) of a single bleb (n bleb = 1, n t = 201). H: Local alignment C ( r 0 ) (black, mean ± s.d.) and correlation length C ( r ) (blue, mean ± s.d.) along bleb length, averaged over the width, time, and several blebs (N = 3, n bleb = 20, n t = 2120). I: Left, Representative high numerical aperture TIRF time-lapse images of 3 μm-confined HeLa ActB -GFP cells. Scale bar, 10 μm. Right, time-lapse images of a bleb front region.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Transfection

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Left panels, representative hNA TIRF image of 3 μm-confined HeLa lifeact-mCherry (black LUT in merge) MYH9 -eGFP cells (fire LUT in merge). Arrows, myosin clusters of various sizes. Scale bar, 10μm. Right panels, zoom of the middle, non-contractile bleb region, in lifeact-mCherry (top, Lifeact, black LUT in merge) and MYH9 -eGFP channels (middle, NMIIA, red LUT in merge). Scale bar, 1 μm. B: Schematic representation of the cortical states found spatially organized in stable blebs: ‘gas-like’ front (I), ‘solid-like’ intermediate region (II), and contractile rear (III). Magenta, actin filaments. Cyan, NMIIA, myosin II filaments. C: Representative high numerical aperture TIRF time-lapse images of 3 μm-confined HeLa ActB -GFP cells. Dashed red polygon, tracking of landmarks (polygon vertices) in the flowing actin network. Dashed back line, bleb contour. Star and arrow, kinks in the bleb contour. Scale bar, 10 μm. D: Bleb contour curvature |κ|, 1/radius (μm -1 , black, mean ± s.d.) and correlation length C(r) (μm, blue, as in panel 3H, mean ± s.d.) along bleb length, averaged over the width and several blebs (N = 3, n bleb = 20).

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques:

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Left, representative high numerical aperture TIRF time-lapse images of HeLa ActB -GFP cells displaying network deformation (black arrows). Scale bar: 10μm (left, whole bleb) and 1μm (right, time-lapse sequence enlarged image from black square). Right, schematic representing the event. B: Left, representative high numerical aperture TIRF time-lapse images of HeLa ActB -GFP cells displaying network rupture (black arrows). Scale bar: 10μm (left, whole bleb) and 1μm (right, time-lapse sequence enlarged image from black square). Right, schematic representing the event. C: Left, representative high numerical aperture TIRF time-lapse images of HeLa ActB -GFP cells displaying membrane-filament snapping (black arrows). Scale bar: 10μm (left, whole bleb) and 1μm (right, time-lapse sequence enlarged image from black square). Right, schematic representing the event. D: Left, representative high numerical aperture TIRF time-lapse images of HeLa ActB -GFP cells displaying membrane-filament retraction (black arrows). Scale bar: 10μm (left, whole bleb) and 1μm (right, time-lapse sequence enlarged image from black square). Right, schematic representing the event. E: Left, non-equilibrium regimes in active systems: active solution (I), pre-stressed gels (II), global contraction (III), and local contraction (IV). Right, Young’s modulus ( E ) is greater than zero in states II and III, where long-range force transmission can occur. Diagram adapted from . F: Top, diagram showing the spatial organization of the three cortical regimes and its correspondence to bleb shape in elongated blebs. Stress percolation: the active solution regime at the bleb front undergoes a stress percolation transition at 〈p〉 > p c to a prestressed gel regime. Failure percolation: the prestressed gel regime undergoes a failure percolation transition at the rear to a global contraction regime, at which . Bottom, diagram showing the spatial organization of the three cortical regimes and its correspondence to bleb shape in blebs displaying a local contraction regime. Coarsening, the active solution regime at the bleb front undergoes a coarsening transition to a local contraction regime, contracting at 〈p〉 < p c . Strain percolation, the local contraction regime undergoes a strain percolation transition at the rear to a global contraction regime, at 〈p〉 > p c . G: Top, fraction of elongated (grey) versus blebs displaying a local contraction regime (cyan) in control (DMSO), 100 μM CK-666 and 40 μM SMIFH2 conditions. Bottom, representative high numerical aperture TIRF images of blebs from HeLa ActB -GFP cells displaying an elongated regime (DMSO, left), or local contraction regime (Local contrac.) under CK-666 (CK-666, middle) and SMIFH2 (SMIFH2, right) conditions. Scale bar, 10 μm. H: Average flow divergence as a function of the average binned actin density. Grey, average for elongated stable blebs (N = 3, n bleb = 20, n t = 2120). Cyan, representative example of a bleb displaying a local contraction regime, corresponding to the SMIFH2-treated bleb displayed in panel 5G. Dashed line, density corresponding to the percolation threshold ( p c = 0.59). Bars, 95% confidence interval. Red region, contraction regime (divergence < 0). Inset, same data plotted as a function of the actin density instead of the binned occupancy. Dashed line, density corresponding to the percolation threshold ( p c = 0.59, Density actin = 0.4).

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Sequencing, Transmission Assay

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Diagram representing the advected percolation model. Parameters are defined in the text. Blue, main cluster. Red, freely-diffusing filaments, not connected to the main cluster. B: Outcomes of the 2D stochastic advected percolation model, phase diagram of the average maximum cluster length along the x-axis, , as a function of the assembly rate β 0 and the advection velocity v 0 . Legend is shown as inset. Parameters are L x = 50 μ m , L y = 10μ m , D a = 10 μ m 2 min -1 , k d = 0.5 min -1 . C: Snapshots of the configurations at different time points for the three different simulations pinpointed in panel 6B (star, percolated regime; triangle, intermittent regime; circle, non-percolated regime). Blue, sites belonging to the cluster attached to the left hand-side boundary. Red, all other sites with a non-zero number of molecules. White, sites without any molecule. Time stamp, simulation time from start. D: Snapshots of the time trajectories of the renormalised maximum cluster length and minimum cluster length for the three different conditions pinpointed in panel 6B (star, percolated regime; triangle, intermittent regime; circle, non-percolated regime). Time, simulation time from start. E: Left, example network analysis of a bleb region, from the actin fluorescence channel (top) to the binarized image (middle), and the extracted skeleton (bottom). Scale bar, 1μm. Middle, representative high numerical aperture TIRF image of HeLa ActB -GFP cells. Scale bar, 10μm. Right, actin network analysis of skeletonized time-lapse images. Blue, labelled C 1 , largest cluster. Red, all other clusters. Time stamp, seconds elapsed from first image in the shown series. Black arrow, region plotted in the histogram in panel 6F. F: Kymograph of the region marked with a black arrow in panel 6E. Blue, largest cluster. Red, all other clusters. Black arrows, stress percolation events (red isolated filaments connect to the main cluster, becoming blue). Scale, 20μm/min G: PIV analysis of the bleb shown in panel 6E at times 0 and 35 s from the start of the acquisition. Squares, front and rear regions used in panels 6H and 6J. Colours, speed magnitude. Scale bar, 10μm. H: Mean PIV Y speed (velocity component parallel to bleb long axis) in the front and rear regions marked in panel 6G, as a function of time from the start of the acquisition (s, seconds). I: Segmented images of the bleb shown in panel 6E at time t = 0s and time t = 35 s. Blue, labelled C 1 , largest cluster. Red, all other clusters. L C1 , maximum main cluster length. J: Renormalised maximum main cluster length L C1 /L as a function of time from the start of the acquisition (s, seconds). Shadow, positions of the front (grey) and rear (cyan) regions from panel 6G-H. K-M: Left, renormalised maximum main cluster length L C1 /L as a function of time from start of acquisition (s, seconds), for three representative bleb examples. Right, cluster analysis of the skeletonized actin network or one representative example. Blue, labelled C1, largest cluster. Red, all secondary clusters. K: Representative examples of persistent phenotype. L: Representative examples of intermittent phenotype. M: Representative examples of retracting phenotype. N: Boxplot of average occupancy 〈p〉 at the bleb front for persistent (white, P), intermittent (light grey, I) and retracting (R, dark grey) blebs. Dashed line, theoretical percolation threshold (p c = 0.59). P , Welch’s T test.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Fluorescence, Isolation

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Outcome of the deterministic advected percolation model with uniform nucleators’ profile. Left, phase diagram of the renormalised maximum cluster length (as shown in the schematics of ) varying the advection speed v 0 and the assembly rate β 0 . Parameters, k d = 0.5 min 1 , D a = 10 μm 2 min -1 , L x = 50 μm, c * = 1 ⊓M. The details of the numerical integration are reported in the Supplementary Information. Inset, legend. Right, renormalised cluster length, L c /L x as a function of elapsed simulation time, for three representative examples pinpointed in the left panel (star, percolated regime; triangle, intermittent regime; circle, non-percolated regime). B: Left, renormalised cluster length, L c /L as a function of simulation time. Parameters are the same as in panel A, other than β 0 = 100 nM/min , v 0 = 1 μ m/min and c * = 0.98 · β 0 / k d · e - L x /2 L N . Right, concentration profile of actin along the bleb length at t = 20 min. The details of the numerical integration are discussed in the SI. C: Left, phase diagram of the stochastic advected percolation model as a function of the advection speed v 0 and the assembly rate β 0 . Legend is shown as inset. Middle, representative dynamics of the renormalised cluster length for the three cases pinpointed in the left panel. Time, elapsed simulation time. Right, lattice configurations for the three cases plotted in the middle panel. Time, elapsed simulation time. Blue, sites belonging to the cluster. Red, free filaments. White, empty sites. D: Colour-coded time projection of contours of four representative persistent, winding, unstable, and retracting blebs from 3 μm-confined HeLa MYH9 -eGFP Lifeact-mCherry cells. Inset, time-colour correspondence. Representative E: Left, fluorescence confocal spinning disk time-lapse images of four representative persistent, winding, unstable, and retracting blebs from HeLa MYH9 -eGFP (NMIIA, first column) lifeact-mCherry cells (Actin, second column), as shown in panel S4D. Black arrows, sites of transient actin cortex reformation. Magenta arrows, intensity line scans plotted in right panel. Scale bar, 10 μm. Right, normalized actin density line scans from magenta arrows in left panel. Persistent blebs (top row) display a leading edge persistently depleted of actin. Intermittent blebs could display a winding front protruded by membrane tearing waves (with actin cortex scars corresponding to the accumulation of percolated actin filaments at alternating sides) (black arrows on “winding” row). Intermittent blebs could also display a more unstable protrusion if a full cortex was reformed, leaving actin cortex scars across the full bleb width (black arrows on “unstable” row). F: Representative fluorescence confocal spinning disk time-lapse images of blebs transitioning between regimes from 3 μm-confined HeLa ActB -GFP (top row) or HeLa MYH9 -eGFP Lifeact-mCherry cells (middle and bottom rows). Time, time elapsed from first frame in seconds. Black arrows, sites of transient actin cortex reformation. Scale bars, 10 μm.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Representative fluorescence confocal spinning disk time-lapse images of a migrating cytoplast from 3μm-confined HeLa ActB -GFP cells (Actin-GFP) at bottom, middle and top planes. Arrow, sparse actin network at the front. Scale bar, 10 μm. B: Representative fluorescence confocal spinning disk time-lapse images of a migrating cytoplast from 3μm-confined HeLa ActB -GFP cells (Actin-GFP middle plane) under adhesive conditions (polylysine coating). Top, zoom on the front cortical region marked with a back square in bottom panel. Arrows, rebuilding of the cortical actin network. Time stamp, time elapsed from first frame in seconds. Bottom, full cytoplast. Scale bar, 10 μm. Right, kymograph of the front of the cytoplast shown in left panels. Scale bars, 5 μm, 5 s. C: Normal edge velocity as a function of the binned actin density of the cytoplast shown in panel 7A, calculated locally for 300 frames (150 s). Colours represent the three observed cortical regimes (yellow, protrusion; orange, stalling; red, retraction). D: Average normal edge velocity as a function of the binned actin density. Black solid line, average for all timepoints of all cytoplasts (n = 16). Coloured light lines, average for all timepoints for each individual cytoplast separately. Dashed black line, predicted percolation transition point, where velocity = 0. Bars, 95% confidence interval. E: Average actin density as a function of the binned normal edge velocity. Black solid line, average for all timepoints of all cytoplasts (n = 16). Coloured light lines, average for all timepoints for each individual cytoplast separately. Dashed black line, predicted percolation transition point, where velocity = 0. Bars, 95% confidence interval. F: Average curvature (μm -1 , 1/radius) as a function of the binned normal edge velocity. Black solid line, average for all timepoints of all cytoplasts (n = 16). Coloured light lines, average for all timepoints for each individual cytoplast separately. Dashed black line, predicted percolation transition point, where velocity = 0. Bars, 95% confidence interval. G: Left, representative fluorescence confocal spinning disk image of a migrating cytoplast from 3μm-confined HeLa ActB -GFP cells (Actin-GFP) at middle plane. Scale bar, 10 μm.vRight, diagram representing the three observed cortical regimes (yellow, protrusion “I”; orange, stalling “II”; red, retraction “III”). The transition from the protrusion to the stalling regime occurs at the percolation density p c . The curvature at the stalling phase tends to 0. H: Left, diagram illustrating the polarity index. Accumulation at the rear yields a polarity index of +1, whereas homogeneous accumulation yields a polarity index of 0. Right, protrusion polarity index as a function of the binned actin polarity index (mean ± s.d., n = 17 cytoplasts and 4952 time points). I: Average persistence time (decay length of the edge velocity spatial autocorrelation averaged over all time points for a given fragment) as a function of the actin-velocity correlation (Pearson’s r ). J: Representative fluorescence confocal spinning disk time-lapse images of a migrating cytoplast from 3μm-confined HeLa ActB -GFP (Actin-GFP, cyan) cells embedded in a collagen gel (Collagen, gray). Time stamp, time elapsed from first image in seconds. Scale bar, 10 μm. K: Average front protrusion (left, μm/min) and actin flow (right, μm/min) of cytoplasts migrating within a collagen get (grey, n = 50) or between two PLL-coated PDMS confinement slides (cyan, n = 79). Cross, mean values. Bars, median values. P values, Welch’s T test. L: Front protrusion (grey line) and retrograde actin flow (cyan line) as a function of time elapsed from first image in seconds, for a representative cytoplast shown in panel 7J. Note the slippage occurring between t = 100 s and t = 180 s. M: Colour-coded time projection of contours of a representative cytoplast shown in panel 7J. Time stamp, time elapsed from first image in seconds. Scale bar, 10 μm. N: Diagram of cortex properties during confined migration. Top left, schematic representing a confinement experiment in a free chamber. Top right, diagram representing an image sequence of confined migrating cell. Pink, mesh representing the actomyosin cortex. Bottom left, schematic representing a confinement experiment in a chamber filled with collagen. Blue, collagen fibres. Bottom right, diagram representing an image sequence of a confined migrating cell inside a chamber filled with collagen. Pink, mesh representing the actomyosin cortex. Blue, collagen fibres. Red arrows, forces exerted on the collagen gel by the migrating cell.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Fluorescence, Migration, Sequencing

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Pipeline for protrusion and actin cross-correlation analysis of bleb fragments. First, the signal is acquired on actin-GFP. The high background of the actin-GFP signal allows for a good segmentation, which is performed manually for a few time points and then fed into Ilastik to segment the rest of the movie. The Fiji plugin ADAPT (Automated Detection and Analysis of ProTrusions) allows for the analysis of protrusion edge and image segmentation. The actin cortical density is calculated on a 300 nm area beneath the cell contour, on background-subtracted actin-GFP movies. The cross-correlation analysis, normalization, and plotting is performed using a homemade Python algorithm. The output of the pipeline is three cross-correlation matrices: edge velocity ⍰ edge velocity, actin ⍰ edge velocity, and dactin/dt ⍰ edge velocity. B: Colour-coded time projection of cytoplast contours and fluorescence confocal spinning disk images of representative migrating cytoplasts from 3μm-confined HeLa ActB -GFP cells (Actin-GFP middle plane) under adhesive conditions, displaying persistent (top), winding (middle), and unstable (bottom) migration. Inset, colour code. C: Cross-correlation (sliding dot product) matrices of edge velocity ⍰ edge velocity, actin ⍰ edge velocity, and d actin /d t ⍰ edge velocity for representative examples of persistent, winding, and unstable migration. Inset, colour scale bar, corresponding to correlation coefficient. D: Top, edge velocity spatial autocorrelation. Black line, mean ± s.d. (n = 17). Coloured lines show edge velocity autocorrelation curves of three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. Bottom, decay lengths of the average curve (grey) and three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. E: Top, edge velocity temporal autocorrelation. Black line, mean ± s.d. (n = 17). Coloured lines show edge velocity autocorrelation curves of three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. Bottom, decay lengths of the average curve (grey) and three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. F: Top, actin ⍰ edge velocity temporal cross-correlation. Black line, mean ± s.d. (n = 17). Coloured lines show cross-correlation curves of three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. Bottom, values of actin-velocity correlation coefficient of average curve (grey) and three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. G: Top, d actin/dt ⍰ edge velocity temporal cross-correlation. Black line, mean ± s.d. (n = 17). Coloured lines show cross-correlation curves of three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. Bottom, minima and maxima near the vertical axis of the average curve (grey) and three representative persistent (green), winding (yellow), and unstable (orange) cytoplasts. H: Representative fluorescence confocal spinning disk time-lapse images of 3μm-confined HeLa ActB -GFP (Actin-GFP) cells embedded in a collagen gel (Collagen). Black arrow, fragmenting bleb. Scale bar, 10 μm. I: Histogram of projected area of 3μm-confined cytoplasts (top row) or stable blebs (bottom row) embedded in 2mg/ml collagen (grey, left column) or in a PLL-coated chamber (cyan, right column). J: Representative fluorescence confocal spinning disk image of a cytoplast from 3μm-confined HeLa ActB -GFP (Actin-GFP, cyan) cells embedded in a collagen gel (Collagen, gray), and collagen deflection map, resulting from comparing with PIV the collagen channel during and after cytoplast migration. Arrow colours, deflection magnitude. Inset, colour scale bar (Deflection, μm). Cyan, actin channel. Grey, collagen channel. K: Left, mean collagen deflection map as in panel S5J (n = 18). Arrow colours, deflection magnitude. Grey, cytoplast contours. Inset, colour scale bar (Deflection, μm). Right, divergence of the mean collagen deflection map shown on the left. Inset, colour scale bar (sign of the divergence). L: Left, representative collagen deflection map during fragment migration. Arrow colours, deflection magnitude. Cyan, actin channel. Grey, collagen channel. Right, diagram showing inferred forces applied to the collagen matrix.

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques: Fluorescence, Migration

Journal: bioRxiv

Article Title: Advected percolation in the actomyosin cortex drives amoeboid cell motility

doi: 10.1101/2022.07.14.500109

Figure Lengend Snippet: A: Left, schematic representing the event of membrane snapping. Middle, representative high numerical aperture TIRF images of HeLa ActB -GFP cells with an event of membrane deformation and snapping. Scale bar, 10μm. Right, zoomed time-lapse images of membrane deformation and snapping (measured angle marked on the image). Time stamp, time elapsed from first frame in seconds. Scale bar, 1μm. B: Bleb membrane snapping angle (°) in control (grey, n = 15) and ezrin inhibitor NSC668994 10μM (orange, n = 19) conditions. P value, Welch’s T test. Cross, average. Bar, median. C: Left, schematic representing the event of membrane tearing. Middle, representative high numerical aperture TIRF images of HeLa ActB -GFP cells with an event of membrane tearing. Scale bar, 10μm. Right, zoomed time-lapse images of membrane deformation (measured angle marked on the image). Time stamp, time elapsed from first frame in seconds. Scale bar, 1μm. D: Bleb membrane tearing angle (°) in control (grey, n = 103) and ezrin inhibitor NSC668994 10μM (orange, n = 66) conditions. P value, Welch’s T test. Cross, average. Bar, median. E: Cytoplast membrane tearing angle (°) in control (grey, n = 39) and ezrin inhibitor NSC668994 10μM (orange, n = 13) conditions. P value, Welch’s T test. Cross, average. Bar, median. F: Left, time-prevalence of unstable (orange), winding (yellow), persistent (green) phenotypes in control and ezrin inhibitor conditions in blebs (control, n = 176 blebs; NSC668994, n = 68) and cytoplasts (control, n = 17 blebs; NSC668994, n = 18). Right, representation of cytoplast blebbing regimes (retracting, unstable, winding, persistent) in control and ezrin inhibitor NSC668994 10μM conditions over 350 frames. G: Average actin polarity in control (grey, n = 17) and ezrin inhibitor NSC668994 10μM (orange, n = 18) conditions. P value, Welch’s T test. Cross, average. Bar, median. H: Retrograde cortical flow or protrusion speeds in control (grey, n = 17) and ezrin inhibitor NSC668994 10μM (orange, n = 18) conditions. P value, Welch’s T test. Cross, average. Bar, median. I: Left, actin cortical gradient from the bleb tip in control (grey, mean ± SD, n = 17), and ezrin inhibitor NSC668994 at 10μM conditions (orange, mean ± s.d., n = 18). Right, d-value (from Kolmogorov–Smirnov test) comparing control and ezrin inhibitor conditions at every point. White area, significant difference (alpha < 0.05). J: Left, cortical actin density in cortical regions flowing from the front to the rear, as a function of time (time=0 is bleb front). Curves are normalized by their plateau. Slope of the curves from normalized density=0.2 to 0.8. P value, Welch’s T test. Right, actin assembly slope (assembly rate). P value, Welch’s T test. Bar, median. K: Left, edge velocity spatial autocorrelation function for control (grey, mean ± s.d., n = 17), and ezrin inhibitor NSC668994 at 10μM conditions (orange, mean ± s.d., n = 18). Right, decay lengths of edge velocity spatial autocorrelation. P value, Welch’s T test. Cross, average. Bar, median. L: Left, edge velocity temporal autocorrelation function for control (grey, mean ± s.d., n = 17), and ezrin inhibitor NSC668994 at 10μM conditions (orange, mean ± s.d., n = 18). Right, decay lengths of edge velocity temporal autocorrelation. P value, Welch’s T test. Cross, average. Bar, median. M: Top, actin ⍰ edge velocity temporal cross-correlation for control (grey, mean ± s.c., n = 17), and ezrin inhibitor NSC668994 at 10μM conditions (orange, mean ± s.d., n = 18). Bottom, actin-velocity Pearson’s correlation coefficient ( r , mean ± s.d.). N: Top, dactin/dt ⍰ edge velocity temporal cross-correlation for control (grey, mean ± s.d., n = 17), and ezrin inhibitor NSC668994 at 10μM conditions (orange, mean ± s.d., n = 18). Bottom, minimum and maximum around Δt = 0 (mean ± s.d.).

Article Snippet: Human cervical adenocarcinoma cells HeLa-Kyoto stably expressing myosin IIA (Myh9)-GFP and LifeAct-mCherry or Myh9-GFP, or LifeAct-mCherry and the plasma membrane-targeting CAAX box fused to GFP, or TALEN-edited ActB fused with GFP (Cellectis, Paris, France), or with no stable marker were cultured in DMEM GlutaMAX medium (Gibco, #61965-026) supplemented with 10% FBS (Gibco, #P30-193306A) and Penicillin Streptomycin (Thermofisher, #15140-122) at 37°C and 5% CO2.

Techniques:

Journal: Oncotarget

Article Title: Modulation of estrogen related receptor alpha activity by the kinesin KIF17

doi: 10.18632/oncotarget.18104

Figure Lengend Snippet: A. Sequence of the KIF17-Tail. The 12 amino acid NR box peptide containing the LXXLL motif is underlined. B. Co-immunoprecipitation of GFP-ERR1 with myc-EV control, myc-KIF17-T or myc-KIF17T ΔNR expressed in HEK293 cells. Immunoprecipitates and total lysates were analyzed by immunoblot using anti-GFP and anti-myc IgG. C. , D. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in MCF7 cells expressing ERRE-Luc and myc-EV, myc-KIF17-T, myc-KIF17 ΔNR (panel C) or myc-KIF17 NR (panel D). E. Luciferase reporter assays showing transcriptional activity of endogenous ERR1 in parental LM2 and LM2 NR cells transfected with ERRE-Luc. F. Same as in panel E, but comparing cells transfected with control or ERR1 siRNAs. Graphs show normalized luminescence values pooled from ≥ 3 experiments performed in triplicate. Error = SEM. *** p < 0.05.

Article Snippet: Lysates were cleared using Protein-G sepharose beads (GE Healthcare) and incubated overnight with 6-8μg rabbit anti-GFP antibody (Novus Biologicals, NB 600-303) or mouse anti-myc antibody (Sigma M4439).

Techniques: Sequencing, Immunoprecipitation, Control, Western Blot, Luciferase, Activity Assay, Expressing, Transfection

Journal: PLoS ONE

Article Title: Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

doi: 10.1371/journal.pone.0054764

Figure Lengend Snippet: A) LacZ positive P3 cells were injected into the digit tip of SCID mice 1 day prior to amputation and collected at 10 DPA when the regenerate at the blastema stage. LacZ positive cells are present at the injection site in the dorsal connective tissue (*) and are scattered throughout the blastema. B) During the differentiation stage (16 DPA) LacZ positive P3 cells are primarily found in the regenerating connective tissue with small clusters of cells present within the trabeculae of the regenerating bone (arrows). C–E) GFP+ human breast cancer cells injected into P3 connective tissue prior to amputation remained aggregated in the regeneration stump and did not enter the blastema. GFP positive cells (C) shown aggregated in the stump of a 16 DPA regenerate indicating that they survive engraftment. Immunohistochemical co-staining for the endothelial marker vWF (D, E) indicates that these cells differentiate in situ without participating in the regenerative response. A and B, scale bar = 200 µm; C–E, scale bar = 100 µm.

Article Snippet: Labeled cells were identified based on immunohistochemical staining with a GFP antibody (Novus biologicals).

Techniques: Injection, Immunohistochemical staining, Staining, Marker, In Situ

Journal: PLoS ONE

Article Title: Connective Tissue Fibroblast Properties Are Position-Dependent during Mouse Digit Tip Regeneration

doi: 10.1371/journal.pone.0054764

Figure Lengend Snippet: A–C) GFP and Ki67 co-immunohistochemical reveal P3 isolated cells engraft and proliferate. A) GFP labeled P3 cells are identified within the digit. B) Ki-67, a marker for proliferation, is expressed in engrafted and endogenous P3 cells. C) Merged GFP and Ki67 expression identifies a fraction of engrafted P3 cells that are proliferating. Double labeled cells shown in C are indicated with arrows in the image set. GFP- red, Ki67- green, DAPI nuclear stain- blue. Scale bar = 50 µm. D) Quantification of the proliferation indices of P2 and P3 engrafted cells compared to endogenous neighboring cells. Left: In unamputated digits 17 days post engraftment, P3 cells proliferated at a rate similar to neighboring control cells, whereas P2 cells are non-proliferative. Right: P3 cells participating in blastema formation at 10 DPA display a proliferation index that was lower than neighboring endogenous cells but significantly higher than P2 cells within the blastema. All chart values are expressed as means ± SEM. The proliferative index of P3 cells is significantly greater than P2 cells in both studies (**, p-value <0.005).

Article Snippet: Labeled cells were identified based on immunohistochemical staining with a GFP antibody (Novus biologicals).

Techniques: Immunohistochemical staining, Isolation, Labeling, Marker, Expressing, Staining, Control