Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Western blots showing the expression levels of IF proteins in astrocytes (two sets) after 3 days of nucleofection with indicated siRNA. Graphs on the right show quantifications from three independent blots, normalised to si ctl and loading control. B) Immunofluorescence images showing Nestin (mouse Ab, magenta), GFAP (rabbit Ab, green) and vimentin (goat Ab, blue) in migrating astrocytes. The white dotted line represents the wound. C) Immunofluorescence of astrocytes stained for vimentin (green) and nuclei (cyan). The white dotted line represents the wound and examples of cell outlines. Quantifications show the spread area of cells during migration. D) Graph of FSC (Forward Scatter) proportional to the cell size, of migrating and non migrating cells in FACS analysis. E) Quantification of cell velocity, persistence and directionality after 24h of migration of astrocytes silenced for a second set of IF proteins (si triple IF set B). F) Immunofluorescence images of centrosome orientation in migrating astrocytes stained for nuclei (cyan) and centrin (red). The white dotted line represents the wound. White arrowheads represent polarised centrosomes, while yellow ones are non polarised. Scale bar 10 μm. The histogram shows the quantification of centrosome polarity (mean ± s.e.m.). Centrosomes are scored as polarised when they are found in the quadrant in front of the nucleus and facing the wound (right scheme). Random orientation of the centrosome with respect to the wound edge corresponds to a value of 25%. Cells analysed must be in the first row and must have neighbour cells on both sides. Data is from N = 3 independent experiments. The sample size for each repeat is: si ctl 30, 37, 50 and si triple IF 30, 37, 50 for S1C; si ctl 49, 49, 49 and si triple IF 50, 50, 50 for S1E; si ctl 137, 247, 150 and si triple IF 222, 200, 151 for S1E. P values are * for p < 0.05, **: p for p < 0.01 and *** for p < 0.001. Scale bar 10 μm.

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Migration

Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Phase contrast images of astrocyte (shown in Movie 1 ) wound healing after 24h of migration. Black lines represent the initial position (0h) and white lines show the final position (24h) of the leading edge. B) Simplified method for calculating persistence and directionality of migration of a cell with nuclear tracking. For more detailed formulas, see Methods section. C) Graphs of cell velocity, directionality and persistence measured by manual nuclear tracking of leader cells after 24h of migration. D) Simplified protocol for plating cells into PDMS rectangular stamps onto hydrogels. The black dotted arrows show the main directions of migration. The central image shows a monolayer of cells migrating on a hydrogel embedded with fluorescent beads (green dots) with representative tractions (T, blue arrows). The last image represents schematically the components of tractions (T x and T y ) analysed with TFM. E) Phase contrast images of astrocytes migrating on a 9kPa collagen-coated polyacrylamide hydrogel at different time points. The white dotted line represents the leading edge, while the arrows show the direction of migration. See also Movie 2 . F) Tractions in the x direction (T x ) at indicated time points. See also Movie 3 . G) Representative kymographs of total tractions (|T|). H) Graphs of tractions in the x direction (T x ), average values (left), values at the edge of the monolayer (middle) and at the centre (right). I) Graph showing the ratio of the tractions at the edge over tractions at the centre, plotted as a function of time of migration. Data is from N = 3 independent experiments. The sample size for each repeat is: si ctl 50, 50, 50, si triple IF 50, 50, 50, si GFAP 50, 50, 50, si Vimentin 50, 50, 49, si Nestin 50, 50, 50 cells for 1C; 2, 1 and 4 videos of si ctl and 2, 3, 3 videos of si triple IF for 1E-I. Graphs show mean ± s.e.m. P values are * for p < 0.05, **: p for p < 0.01 and *** for p < 0.001. Scale bar 50 μm.

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Migration

Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Immunofluorescence images of actin fibres in migrating astrocytes stained for nuclei (cyan) and actin (phalloidin, grey). B) Graph showing the percentage of leader cells that present interjunctional transverse arcs (ITAs) in si control (si ctl) and IF-depleted cells (si triple IF). C) Rose plot showing the frequency of angle distribution of actin fibres analysed in follower cells . Fibres with a 0° angle are perpendicular to the wound while fibres with an angle close to 90° are parallel to the wound. D) Schematics showing the position of the kymographs acquired in E-G . E) Frames of migrating si ctl and si triple IF astrocytes transfected with LifeAct-mCherry. The white dotted line represents the wound while the pink dotted lines represent the positions in which the kymographs were calculated. Kymographs (fire LUT) show the retrograde flow of actin on cell-cell junctions over a time period of 4h. The graph shows the mean retrograde flow speed of actin cables measured at the level of the cell-cell junctions. F) Frames from Movie 4 of migrating astrocytes transfected with GFP-MRLC. The thick white dotted line represents the outline of nearby cells. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymograph indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of myosin at the front and at the rear of myosin longitudinal fibres over a time period of 15 min. The graph shows the mean speed of the myosin retrograde flow at the cell front and at the rear of the longitudinal fibre calculated from the kymographs. The white dotted lines indicate the position of the wound. G) Immunofluorescence images from Movie 5 showing GFP-N-cadherin expressing astrocytes. Insets show the corresponding phase-contrast image. ‘N’ indicates the nucleus. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymographs indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of N-cadherin over a time period of 2h. The graph shows the mean retrograde flow speed of the N-cadherin flow in si ctl and si triple IF migrating astrocytes. H) Staining for N-cadherin (grey) and nuclei (cyan) in migrating astrocytes nucleofected with the indicated siRNAs. Histogram shows the mean percentage ±s.e.m of continuous junctions between adjacent leader cells. White dotted line indicates the position of the wound. Data is from N = 3 independent experiments. The sample size for each repeat is: si ctl 64, 67, 92 and si triple IF 44, 96, 122 for 2B; si ctl 11, 10, 8 stacks and si triple IF 12, 10, 8 stacks for 2C; si ctl 30, 40, 32 and si triple IF 18, 33, 30 for 2E; si triple IF f 22, 27, 18 and si triple IF b 22, 24, 20 for 2F; si ctl 14, 38, 28 and si triple IF 38, 49, 14 for 2G; si ctl 104, 120, 178 si GFAP 134, 137, 125, si vimentin 113, 180, 133, si nestin 132, 190, 123 and si triple IF 122, 225, 112 for 2H. P values are *** for p < 0.00. Scale bar 10 μm for all images except kymographs for which scale bar is 5 μm.

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Immunofluorescence, Staining, Transfection, Expressing

Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Immunofluorescence images of migrating astrocytes showing paxillin (magenta) for focal adhesions, cadherin (yellow, far red) for adherens junction, actin (phalloidin, green) and nuclei (cyan). B) Immunofluorescence images of migrating astrocytes nucleofected with the second set of si triple IFs (set B) and stained for nuclei (cyan) and F-actin (phalloidin, white). C) Immunofluorescence images of actin fibres in migrating astrocytes nucleofected with the indicated siRNAs and stained for F-actin (phalloidin, white). Graph shows the percentage of leader cells that present interjunctional transverse arcs (ITAs) in si control (si ctl) and si single depleted cells (si GFAP, Vimentin or Nestin). D) Maximal intensity projection of migrating astrocytes stained for nuclei (cyan) and actin (phalloidin, white) to show the orientation of actin in the rows behind the leading edge and used for the analysis of . E) Immunofluorescence images of migrating astrocytes nucleofected with the second set of si triple IFs (set B) and stained for nuclei (cyan) and N-cadherin (grey). F) Staining for N-cadherin (grey) and nuclei (cyan) in migrating astrocytes nucleofected with the indicated siRNAs compared to si ctl and si triple IF in . In all images, white dotted line indicates the position of the wound. Data is from N = 3 independent experiments. The sample size for each repeat is: si ctl 71,145, 136, si GFAP 61, 131, 142, si vimentin 60, 135, 142 and si nestin 100, 131, 130 for S2C: si ctl 104, 120, 178 si GFAP 134, 137, 125, si vimentin 113, 180, 133, si nestin 132, 190, 123 and si triple IF 122, 225, 112 for S2F. P values are * for p < 0.05, **: p for p < 0.01 and *** for p < 0.001. Scale bar, 10 μm

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Fluorescence SIM-3D images of a migrating astrocytes transfected with GFP-vimentin and paxillin-Orange shown in Movie 6 (see also Movie 7 ). The orthogonal projections show that vimentin and paxillin are found in the same focal plane. B) Immunofluorescence images of migrating astrocytes stained for nuclei (cyan) and paxillin (green). The white dotted lines indicate the position of the wound. Insets show enlarged images of focal adhesions at the leading edge and in a central region of the cell. C) The top left graph shows the mean number of focal adhesions per cell and the bottom left graph show the mean area of focal adhesions. Adhesions were projected on the nucleus-tip axis (see schematics). The central top graph shows the normalised distance to the nucleus centre of each FA. The central bottom graph shows the distribution of adhesions along the nucleus-tip axis. D) Lifetime of GFP-paxillin positive adhesions in migrating leader astrocytes (top). Duration time of assembly and disassembly of these adhesions (bottom). E) Lifetime of GFP-paxillin positive adhesions in migrating astrocytes (left) of the second and third rows. Duration time of assembly and disassembly of these adhesions (right). F) Immunofluorescence images of astrocytes in the migrating monolayer stained for nuclei (cyan) and paxillin (green). Data is from N = 3 independent experiments. The sample size for each repeat is: si ctl 49, 50, 50, si triple IF 50, 50, 50 and si plectin 50, 50, 55 for 3C; si ctl 26, 40, 40 and si triple IF 26, 40, 40 for 3D, si ctl 16, 16, 35 and si triple IF 8, 36, 37 for 3E. P values are n.s. (not significant) for p > 0.05, * for p < 0.05, **: p for p < 0.01 and *** for p < 0.001. Scale bar 5 μm for 3A and 10 μm for 3B and 3F.

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Fluorescence, Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

doi: 10.1101/328609

Figure Lengend Snippet: A) Immunofluorescence images of migrating astrocytes nucleofected with control siRNA (si ctl) or with a second set (set B) of si triple IFs and stained for paxillin (green) and nuclei (cyan). B) Immunofluorescence images of migrating astrocytes nucleofected with the indicated siRNAs and stained for talin (green) and nuclei (cyan). C) Immunofluorescence images of migrating astrocytes nucleofected with the indicated siRNAs and stained for tubulin (green), centrin (magenta) and nuclei (cyan). D) Temporal colour-code projection of EB3-GFP movies acquired on a confocal spinning disk (one image/5 s). Plots show mean velocity, persistence and directionality of EB3 comets compared to the wound. Histogram (mean ± s.e.m) shows the percentage of EB3 comets that have a positive directionality towards the wound. E) Western blot showing the expression level of plectin upon nucleofection with si plectin compared to si ctl. Histogram (mean ± s.e.m) shows the quantifications from three independent blots, normalised to si ctl and loading control. F) Immunofluorescence of migrating astrocytes stained for plectin (green) and vimentin (magenta). The sample size for each repeat is: si ctl 1650, 1108, 1378 and si triple IF 1652, 1035, 1331 (from at least 6 cells for experiment) for S3D. P values are n.s. (not significant) for p > 0.05, * for p < 0.05 and *** for p < 0.001. Scale bar 10 μm

Article Snippet: DNA plasmids used were: pEGFP-paxillin (gift from E. M. Vallés, Institut Curie, Paris), pPaxillin psmOrange (Addgene 31923), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI (gift from M. Piel, IPGG, Paris), pEGFP-N3-Vimentin , mCherry-N3-Vimentin cloned from pEGFP-N3- Vimentin, pVimentin psmOrange (Addgene 31922), pLifeAct-mCherry (gift from M. Piel, IPGG, Paris), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, CRBM, Montpellier) eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie, Paris), vinculin tension sensor (Vin TS, Addgene 26019 ( )) and mEB3-FL pEGFP-N1 (gift from N. Morin, CRBM, Montpellier).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes

doi: 10.1371/journal.pone.0136952

Figure Lengend Snippet: (A-C) Decreases in the levels of N-cadherin and desmoglein1/2 in HL-1 cells during ethanol exposure. Cells were incubated in medium containing 2% ethanol for the indicated time periods, and the N-cadherin and desmoglein1/2 protein levels were determined by western blot analysis. Hsc70 served as a loading control (A). Quantitative analysis of N-cadherin (B) and desmoglein1/2 (C) is shown. Each graph shows mean±S.E. (n = 3). **, p<0.01, *, p<0.05 versus 0 h. (D) Rupture of the organization of myofibrils and intermediate filaments in ethanol-exposed HL-1 cells. Cells were incubated in medium containing 2% ethanol for 24 hours, stained with phalloidin-rhodamine to visualize filamentous actin, and observed under a confocal fluorescence microscope (upper panel). Cells were also transfected with the vimentin-PSmOrange expression vector, exposed to 2% ethanol for 24 hours, and observed under a fluorescence microscope (lower panel).

Article Snippet: Plasmid vectors expressing Cx43-mApple or mGFP-Cx43 (provided by Dr. Mattias M. Falk, Lehigh University) [ ], LAMP1-mGFP (provided by Dr. Esteban C. Dell'Angelica, University of California Los Angeles) [ ], LAMP1-RFP (Provided by Dr. Walther Mothes, Yale University) [ ] (Addgene plasmid 1817), LMP2-GFP (provided by Dr. Jacques Neefjes, The Netherlands Cancer Institute) [ ], GFP-Cx43Y286A (provide by Dr. Henrique Girao, University of Coimbra, Portugal) [ ], and vimentin-PSmOrange (provided by Dr. Vladislav Verkhusha, Albert Einstein College of Medicine) [ ] (Addgene plasmid 31922) were mixed with Lipofectamine2000 (Invitrogen) and added to the medium, and the samples were incubated overnight.

Techniques: Incubation, Western Blot, Staining, Fluorescence, Microscopy, Transfection, Expressing, Plasmid Preparation