Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: ( A-B ) Metascape enrichment network visualization showing the intra-cluster and inter-cluster similarities of enriched terms (up to ten terms per cluster), in starvation ( A ) and FCS stimulation state (B) separately, where nodes are represented by pie charts indicating their associations with three different mutants. The colour code for pie sector represents G12D (red), G13D (blue), and Q61H (green). Cluster labels were added manually. ( C - D ) Heatmap showing the log2(FC MUT/WT) of differentially enriched proximal proteins which were reported in previous proximitome studies ( C ) or proposed based on our study ( D ) Asterisk indicates that the hit meets the criteria of log2(mutant/WT FC) > 0.5 or <-0.5 and - log10 (p value) > 0.7. ( E ) Heatmap showing the relative abundance (log2 LFQ) of proximal proteins involved in KRAS related canonical pathways (such as MEK-RAF, mTOR, PI3K, RALGDS, RASSF and TIAM RAC pathway).

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Western blot indicates the biotinylated proteins in the presence or absence of different c reagents including tetracycline, phenol-biotin and H 2 O 2 . K-Ras APEX-2 stable cell line was treated with tetracycline for 24 hours where is indicated. Then, cells were treated with phenol-biotin and/ or H 2 O 2 (where is indicated) and cells were lysed, and proteins were separated in an SDS Gel. Streptavidin, DyLight 488 Conjugated antibody were used to visualise the biotinylating proteins. * Represents biotinylated background proteins. (B) Western blot analysis showing a-FLAG in cell extract upon 24 hours tetracycline treatment. (C) Imaging of live cells using Opera Phenix™ and processed in Columbus Image Analysis System. Panels 1183 Hoechst 33342 for staining DNA (blue); the green fluorescence signal from GFP (Green), Differential Interference Contrast (DIC). (D) Western blot analysis showing K-Ras GFP, GAPDH and GFP expression upon digitonin cytoplasmic-membrane fractionation of Stable K-Ras GFP or GFP only FRT T-Rex HEK293 cell line. Cells were incubated with tetracycline for 24 hours. (E) Western blot analysis showing a-FLAG, endogenous KRAS, TOM20 and Calreticulin expression upon digitonin cytoplasmic-membrane fractionation of stable KRAS APEX-2 WT, G12D, G13D and Q61H FRT T-Rex HEK293 cell lines. Cells were incubated with or without tetracycline for 24 hours. (F) Western blot analysis showing a-FLAG, GAPDH, TOM20 and endogenous KRAS in sucrose gradient fractions upon 15 hours of starvation or 15 hours of starvation and a subsequent 10-minute treatment with 20% FCS.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Western Blot, Stable Transfection, SDS-Gel, Imaging, Staining, Fluorescence, Expressing, Membrane, Fractionation, Incubation

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A-F) Volcano plotting of fold changes and p values derived from t-test statistic for proximal proteins identified in G12D, G13D, and Q61H KRAS mutant in either starvation or FCS treated condition, in which WT-KRAS was used as a control. Known KRAS effectors (red), repressors (green) and receptors (blue) are coloured and labelled.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Heatmap showing relative abundance of known KRAS interactors in WT KRAS, G12D, G13D and Q61H KRAS mutant-APEX2 samples in either starvation or FCS stimulated condition. (B) APEX-2 labelling was performed in all different cell lines (WT, G12D, G13D and Q61H) under starvation and FCS induction. Western blot analysis showing ERK, pERK, ARAF and LZTR1 in cell extract as well as elution upon streptavidin-immunoprecipitation. (C) Cells were seeded in 96 well plates and treated with tetracycline for 24 hours. Next day cells were transfected with LZTR1 siRNA or Control siRNA. Cells were imaged after 72 hours using the Opera Phenix microscope. The images were analysed in Columbus Image Data Storage and Analysis System. The media fluorescent per well per mean per cell was determined. Finally, a fold difference between the control and the siRNA LZTR1 was measured. (D) Stable HEK293 FRT T-REX cell lines of WT, G12D, G13D and Q61H were treated with tetracycline for 24 hours. The next day, cells were treated with 50 μg/mL cycloheximide (CHX). At the indicated time points after CHX-treatment cells were harvested. Western blot analysis showing GFP, Pan-Ras and ACTB in cell extract was performed. (E) Band densities from were analyzed using ImageStudio, and expression was normalized to ACTB protein levels to determine KRAS half-life. (F) The influence of LZTR1 overexpression on WT K-Ras as well as G12D, G13D and Q61H oncogenic mutants. HEK293 cells were seeded in 6 well plates and transfected with 1µg Flag-K-Ras plasmids as well 0, 0.1 or 0,25 µg Myc-LZTR1 plasmid concentration. Cells were cultured for a total of 48 hours before protein levels were evaluated using Western blot. (G) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2 µg Myc-LZTR1 plasmid concentration. Cells were incubated for 48 hours, then cells were treated with 50 μg/mL CHX. At the indicated time points after CHX-treatment cells were harvested. Western blot analysis showing FLAG (RAS), MYC (LZTR1) and ACTB in cell extract was performed. (H) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2µg Myc-LZTR1 plasmid concentration and let to grow for 48 hours. Cells were then treated for 24 hours with MLN4924 in different concentrations (0, 0.3 1 µM). Cell extract was then analysed on western blot and FLAG (RAS), MYC (LZTR1), CUL3 and ACTB was monitored. (I) HEK293T cells were transfected with 1 µg Flag-RAS and 0.2 µg Myc-LZTR1 plasmid concentration and let to grow for 48 hours. Cells were then treated for indicated time with MLN4924 (0.3 µM). Cell extract was then analysed on western blot and FLAG (RAS), MYC (LZTR1), CUL3 and ACTB was monitored.

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Mutagenesis, Western Blot, Immunoprecipitation, Transfection, Microscopy, Expressing, Over Expression, Plasmid Preparation, Concentration Assay, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Interplay Between KRAS and LZTR1 Protein Turnover, Controlled by CUL3/LZTR1 E3 Ubiquitin Ligase, is Disrupted by KRAS Mutations

doi: 10.1101/2021.11.23.469679

Figure Lengend Snippet: (A) Microscopy based analysis of GFP-KRAS in the presence and absence of LZTR1. ( B ) KRAS and Pan- Ras protein stability in the presence and absence of LZTR1 assessed by immunoblotting. ( C ) Cullin 3 (CUL3) knockdown stabilises LZTR1. ( D ) LZTR1 (MYC) and KRAS (FLAG) levels are correlated at the protein level. ( E ) KRAS wildtype and G12D (both FLAG tagged) differentially affect LZTR1 (MYC-tag) protein levels. ( F ) KRAS (FLAG) and LZTR1 (MYC) protein levels are dependent on the presence of Cullin 3 (CUL3).

Article Snippet: The stable transfected Flp-In™ T-REx™ 293 Cell Lines (KRas WT-APEX2, KRas G12D -APEX2, KRas G13D -APEX2, KRas Q61H –APEX2, KRas WT-GFP, KRas G12D - GFP, KRas G13D -GFP, KRas Q61H –GFP, GFP only) were cultured in the same medium and selected by adding 100 μg/ml of hygromycin B (InvivoGen, ant-hg-1) to the media.

Techniques: Microscopy, Western Blot

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: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Confirmation of binding affinity between Cntnap4 phages and Nell-1 using a binding dissociation constant ELISA assay. (A) The Basic Local Alignment Search Tool (BLAST) result of the amino acid sequence displayed by the T7 phage constructed with human brain cDNA matched the human Cntnap4 partial protein sequence. Query = the amino acid sequence enclosed by the T7 phage DNA; Sbjct = the matched amino acid sequence Cntnap4. The LamG domains are highlighted in pink. (B) By increasing the number of phages incubated with Nell-1 precoated ELISA plates, the Cntnap4 phage demonstrated significantly higher binding affinity than the control phage. (C) The Cntnap4 phage revealed high binding affinity only to full-length Nell-1 and not to LamG domain-deleted Nell-1. (D) Structures of Nell-1 and Cntnap4 and their potential interaction domains. Nell-1 is a secreted protein composed of 810 amino acids with a molecular weight of ~90 kDa before N-glycosylation and oligomerization. It contains several structural motifs including a laminin G (LamG) domain, a coiled-coil (CC) domain, five cysteine-rich (CR) domains, and six epidermal growth factor (E)-like domains. Cntnap4 is a transmembrane protein of 1310 amino acids consisting of a large extracellular domain, a single membrane-spanning domain, and a short cytoplasmic region at the carboxy-terminus. The extracellular region is composed of a discoidin-like domain (DISC), a fibrinogen-related domain (FreD), two E repeats, and four LamG domains. The cytoplasmic region contains a binding site for PDZ domains. The potential binding domain of Nell-1 and Cntnap4 is highlighted by the blue dashed line. TM= transmembrane. Mean ± SEM of six independent experiments performed in triplicate is shown. *p < 0.05 when compared with control phage.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Construct, Incubation, Molecular Weight

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 and Nell-1 colocalization in osteogenic-committed cells. (A) Of the eight types of tested cell lines, MC3T3-E1 pre-osteoblasts expressed the highest levels of Cntnap4. (B) Of the four types of tested primary cells, NMCC exhibited the highest expression levels of Cntnap4. NMCC = newborn mouse calvarial cells. mRC = mouse rib chondrocytes. hBMSC = human bone marrow stem cells. hARC = human articular chondrocytes. Mean ± SEM of six independent experiments performed in triplicate is shown (A, B). In addition, Nell-1 significantly increased the levels of Cntnap4 in both MC3T3-E1 pre-osteoblasts (C) and NMCC (D). On the contrary, expression of Cntnap2, which was markedly lower than that of Cntnap4, was not responsive to Nell-1 simulation. Mean ± SEM of three independent experiments performed in duplicate is shown (C, D). *p < 0.05 when compared with the group without Nell-1 treatment; #p < 0.05 when compared with the Cntnap4 expression. Moreover, CLSM revealed the colocalization of Nell-1 and Cntnap4 in MC3T3-E1 pre-osteoblasts after 30 minutes of incubation with exogenous recombinant human Nell-1 (E). Colocalization was predominantly found on the plasma membrane. The direct Nell-1/Cntnap4 interaction was validated by Duolink PLA. Similar Nell-1 and Cntnap4 colocalization and protein interactions were also observed in the plasma membrane of NMCC with 30 minutes of Nell-1 treatment (F). Scale bar = 50 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Expressing, Incubation, Recombinant

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 and Nell-1 colocalization in mouse calvarial bone marrow cavities. (A, B) Calvarial bones of 60-day-old mice showed high-intensity double staining of Nell-1 and Cntnap4 inside the bone marrow cavities. White arrows = marrow cavity cells with both Nell-1/Cntnap4 colocalization staining and PLA signaling; yellow arrow = bone lining cells with Nell-1/Cntnap4 colocalization staining. Nell-1 was detected in the calcified bone of 60-day-old mouse calvaria (C) but not in the surrounding muscle (D). However, unlike the marrow cavity where Nell-1 and Cntnap4 colocalize, Cntnap4 was barely detected in the calcified areas of mouse calvarial bone and the surrounding muscle. Scale bar = 500 μm (black), 100 μm (blue), and 20 μm (white).

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Double Staining, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Physical interaction between Nell-1 and Cntnap4. Pull-down assays were performed with MC3T3-E1 pre-osteoblasts (A) and NMCC (B). Increased Cntnap4 was detected when beads were coated with His-tagged Nell-1. Co-immunoprecipitation assay with MC3T3-E1 pre-osteoblasts (C) and NMCC (D) demonstrated an increase in Cntnap4 when cells were incubated with Nell-1. To confirm specificity, no Cntnap4 was detected when the agarose beads were not coated with anti-Nell-1 antibody. (E) SPR assay was performed to assess the binding affinity and dynamic relationship between pentameric Nell-1 (Nell-1(5)) and the immobilized Cntnap4extra, which demonstrated classical ligand-receptor binding. Red dashed lines present the kinetic projection performed by Scrubber 2.0 (BioLogic Software Pty Ltd., Campbell, Australia).

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Co-Immunoprecipitation Assay, Incubation, SPR Assay, Binding Assay, Software

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 is indispensable for Nell-1 osteogenic bioactivity in vitro. (A) ALP staining on day 9 and Alizarin red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA-transfected MC3T3-E1 cells. In Cntnap4-KD MC3T3-E1 cells, high staining intensities of ALP and Alizarin red were only present in the BMP2 group. (B) A time-dependent, steady increase in Ocn and Opn staining was observed in both PBS and recombinant human Nell-1-treated control MC3T3-E1 cells. At each time point, the Nell-1-treated group demonstrated increased staining intensity when compared with the PBS-treated group. In Cntnap4-KD MC3T3-E1 cells, neither PBS nor Nell-1 treatment resulted in detectable positive staining of Ocn or Opn. (C) In Control MC3T3-E1 cells, Alp, Collagen Iα1, and Collagen Iα2 reached peak expression levels 9 days after stimulation, whereas Ocn, Opn, and Bsp displayed time-dependent patterns of increased expression. Notably, higher expression levels were detected for each gene in the Nell-1 treatment group when compared with the PBS group. However, Cntnap4-KD MC3T3-E1 cells did not exhibit significant changes in any osteogenic markers nor any differences between the PBS group and the Nell-1 group. Mean ± SEM of six independent experiments performed in triplicate is shown. *p < 0.05 when compared with the Control + PBS group. Scale bar = 100 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: In Vitro, Staining, shRNA, Transfection, Recombinant, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4-KD blocks the osteogenic effects of Nell-1 ex vivo. (A) Mineral deposition in the mouse calvarial explants was revealed by Alizarin Complexone during the culture period. Lentiviral overexpression of Nell-1 increased the density of Alizarin Complexone; however, when Nell-1 was overexpressed in Cntnap4-KD samples, the Alizarin Complexone staining was comparable to the control (without Cntnap4-KD or Nell-1 overexpression). (B) Quantification of the maximal width of the frontal and parietal bone overlapping area. Nell-1 overexpression alone increased the overlapping area, whereas Cntnap4-KD alone slightly reduced the overlapping area. When the Cntnap4-KD samples were treated with Nell-1 lentiviral overexpression, the maximal width of the overlapping area remained unchanged (similar to that of the control group). (C) Quantification of the unclosed anterior fontanel area. The calvarial explants in the Nell-1 overexpression group demonstrated completely closed fontanels, whereas anterior fontanels in the control group remained open. Cntnap4-KD alone slightly inhibited the closure of the anterior fontanels. The Cntnap4-KD + Nell-1 overexpression group showed a largely open fontanel area. The edge of each calvarial bone is outlined by a white dotted line; yellow arrows represent the maximal width of the frontal and parietal bone overlapping area (in the coronal suture). P=parietal; F=frontal. Eight calvaria explants were used for each group. For B and C, the means were used as center values. The Mann–Whitney test was used for statistical analysis. p < 0.05, p < 0.01. Scale bar = 100 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Ex Vivo, Over Expression, Staining, MANN-WHITNEY

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Wnt1-Cre-mediated Cntnap4-knockout (Cntnap4flox/+;Wnt1-Cre) neonatal mice demonstrate decreased craniofacial development. Representative superior view of gross appearance (A), skeletal staining (B), and 3D micro-CT reconstruction (C) images of the craniofacial skeleton of neonatal Cntnap4flox/+;Wnt1-Cre mouse and its wild-type littermate demonstrate the significant difference in the cranial bone formation and sutural patency. In comparison with its wild-type littermate, the neonatal Cntnap4flox/+;Wnt1-Cre mouse has significantly less bone in the anterior fontanel of calvaria (red asterisk). In addition, defective mineralization and bone formation are also observed in the coronal suture (red arrows) of the Cntnap4flox/+;Wnt1-Cre mouse skull, which can also be appreciated from the lateral view (D) and 3D micro-CT reconstructions (E). Six pairs of neonatal Cntnap4flox/+;Wnt1-Cre mice and their wild-type littermates were compared. F = frontal bone; P = parietal bone; I = interparietal bone; N = nasal bone. Scale bar = 1 mm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Knock-Out, Staining, Micro-CT

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 is indispensable for the Nell-1-responsive activation of MAPK and Wnt signaling pathways in vitro. (A) Activation of MAPK signaling in Control and Cntnap4-KD MC3T3-E1 pre-osteoblasts stimulated with Nell-1. In Control MC3T3-E1 cells, significantly higher levels of pERK and pJNK were detected 10 minutes and 30 minutes after Nell-1 stimulation, respectively. There was no change detected in the phosphorylation level of P38. In Cntnap4-KD MC3T3-E1 cells, Nell-1 stimulation did not alter the expression levels of pERK and pJNK. Expression of Wnt signaling molecules in the whole cell lysate (B) and cell nuclear lysate (C) of Control and Cntnap4-KD MC3T3-E1 cells treated with Nell-1. In Control MC3T3-E1 cells, Nell-1 significantly increased the expression levels of Axin2 and active β-catenin, whereas no effect was observed on these markers in Cntnap4-KD cells treated with Nell-1. The charts demonstrate mean relative band intensity (normalized to control MC3T3-E1 at 0 minutes) ± SEM for three individual experiments. p < 0.05 when compared with the Control group at 0 minutes, #p < 0.05 when compared with the Cntnap4-KD group at 0 minutes.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Activation Assay, In Vitro, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Schematic diagram of Nell-1 signaling pathways during osteogenesis. As a secreted molecule, Nell-1 initiates cellular signaling through binding to its specific receptor, Cntnap4, on the cell surface. The MAPK and Wnt signaling pathways play critical roles in Nell-1-mediated osteogenesis. Nell-1 preferentially activates ERK and JNK in MAPK signaling and also promotes the phosphorylation/activation of Runx2, which stimulates the expression of Nell-1 and Ocn by directly binding to the OSE2 region of their promoters. In addition, Nell-1 promotes the expression of Axin2 and active β-catenin and increases the nuclear translocation of active β-catenin.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Binding Assay, Activation Assay, Expressing, Translocation Assay