Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.201801162

Figure Lengend Snippet: IFs limit the generation of traction forces at FAs. (A) Immunofluorescence confocal images in two distinct focus planes of astrocytes stained for vinculin. The white dotted line indicates the position of the wound. (B) Graph showing the percentage of junctions showing positive vinculin staining. (C) Fluorescence images of migrating astrocytes expressing VinTS (Green Fire Blue LUT). Representative frequency distribution of frequencies of FRET index (central graph) and quantification of the mean FRET index (inversely related to vinculin tension) of vinculin in FAs (right graph). Each dot represents a FA. The white dotted line indicates the position of the wound. (D) Immunofluorescence and intensity projection (fire LUT) of paxillin (magenta) and actin (green) on micropatterned cells. Inset shows the crossbow-shaped micropattern. Projections are calculated on one experiment representative of three. (E) Left: Tractions fields of astrocytes plated on collagen-coated crossbows. Arrows indicate the orientation, color, and length of the local magnitude of the force in Pa. Right: Quantification of traction forces in single micropatterned cells. Data are from n = 3 independent experiments. Histograms show mean ± SEM. The sample size for each repeat: si ctl 137, 247, 150 and si triple IF 222, 200, 151 for B; si ctl 2,537, 3,312, 1,473 adhesions and si triple IF 1,447, 2,548, 2,081 adhesions for C; si ctl 32, 21, 25 and si triple IF 30, 46, 38 for E. ***, P < 0.001. Bar, 10 µm.

Article Snippet: DNA plasmids used were pEGFP-paxillin (gift from E.M. Vallés, Institut Curie, Paris, France), pPaxillin psmOrange (31923; Addgene), pPaxillin-mCherry cloned from pPaxillin psmOrange with BamHI and NotI, pEGFP-N3-vimentin , RFP-N3-vimentin cloned from pEGFP-N3-vimentin with NotI and EcoRI, pVimentin psmOrange (31922; Addgene), pLifeAct-mCherry (gift from M. Piel, Institut Pierre-Gilles de Gennes, Paris, France), pEGFP-N-cadherin (gift from C. Gauthier-Rouvière, Cell Biology Research Center, Montpellier, France), eGFP-RLC Myosin II (gift from B. Latge and B. Goud, Institut Curie), vinculin tension sensor (Vin TS; 26019; Addgene; ), and mEB3-FL-pEGFP-N1 (gift from N. Morin, Cell Biology Research Center, Montpellier, France).

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Generation of stress fibers through myosin-driven re-organization of the actin cortex

doi: 10.1101/2020.06.30.179283

Figure Lengend Snippet: A) TIRF time-lapse imaging of a migrating U2OS cell. The selected time-lapse frames (from the yellow boxed area) illustrate the assembly of a cortical stress fiber (exemplified by white arrows) below the moving nucleus. Histone H2B-mCherry was applied to detect the nucleus (blue, EPI-TIRF) and miRFP670-paxillin (magenta) and LifeAct-TagGFP2 (grey) to visualize focal adhesions and F-actin, respectively. See also Movie S3. B) Quantification of the position of de novo formation of cortical stress fibers from TIRF-time-lapse videos of U2OS cells expressing LifeAct-TagGFP2, Histone-H2B-mCherry, and miRFP670-paxillin. Data are presented as %-distribution of cortical stress fibers assembled under the nucleus versus outside the nucleus perimeter ± SEM. n = 101 cortical stress fibers analyzed from 19 cells. C) Quantification of NMIIA pulse location at the ventral cortex of U2OS cells expressing Histone H2B-eGFP and mApple-NMIIA. Data are presented as pulse frequency (number of individual pulses) under the nucleus vs. outside the nucleus perimeter (rest of the ventral cortex). Each data point is normalized to the area of nucleus. Box plot displays median, outlier whiskers and ‘x’ points out the mean. n= 19 cells. Significance ( p = 0.00012) tested with a paired t -test. D) TIRF time-lapse imaging of a migrating U2OS cell expressing eGFP-NMIIA (green) and vinculin-mApple (magenta). Selected time-lapse frames from the magnified area (yellow box) demonstrate that NMIIA pulse (yellow arrow) is associated with the assembly of cortical stress fibers and enforcement of vinculin positive focal adhesions (orange arrows). See also Movie S2 . Scale bars 10 μm and 5 μm for whole cell images and time-lapse zoom-ins, respectively. Imaging interval 30s.

Article Snippet: LifeAct-TagGFP2 was a kindly provided by the Emmanuel Lemichez lab. Histone H2B-eGFP (in peGFP-N3 backbone) and Histone H2B-mCherry (in pmCherry-N3 backbone) were gifts from Maria Vartiainen lab. miRFP670-paxillin (pPL1514) was constructed by replacing the GFP in eGFP-paxillin (containing gallus gallus paxillin ORF with T132S and M133L unattended mutations in an eGFP-C1 backbone) with miRFP670 originated from miRFP670-N1 (a gift from Vladislav Verkhusha, Addgene plasmid #79987; ) via restriction free cloning using NEBuilder (New England Biolabs).

Techniques: Imaging, Expressing

Journal: bioRxiv

Article Title: Generation of stress fibers through myosin-driven re-organization of the actin cortex

doi: 10.1101/2020.06.30.179283

Figure Lengend Snippet: A) Formation of a cortical stress fiber from the ventral cortex of a migrating U2OS cell as studied by time-lapse TIRF microscopy. The entire cell is shown on right, and the selected time-lapse frames (from the region indicated by yellow box) display how F-actin (grey, LifeAct-mKate2; blue arrows) and NMIIA (green, eGFP-NMIIA; yellow arrows) organize into an actomyosin bundle, which promotes the enlargement of pre-existing paxillin-positive focal adhesions (FA) (magenta, miRFP670-paxillin). Note that prior to bundle assembly, the pre-existing focal adhesions were connected to different actomyosin bundles. Original imaging interval 30s. See also Movie S4 . Scale bars 10 μm and 5 μm for whole cell image and magnified time-lapse frames, respectively. B) Percentual distribution of cortical stress fiber numbers in U2OS cells and MEFs after different pharmacological perturbations. Cortical stress fiber number and length measurements were obtained through blind analysis of the tile-scan TIRF data. n= DMSO 157 and 247, CK-666, 166 and 182, Blebbistatin 195 and 198 cells for U2OS cells and MEFs, respectively. See also Figure S4A and S4C. C) Schematic representation of the de novo cortical stress fiber assembly from the ventral actomyosin cortex. 1. NMIIA pulses occur frequently at the ventral actin cortex. 2. These pulses can cause transient accumulation and bundling of the cortical actin filaments via myosin-mediated actin filament crosslinking and re-organization. This triggers the enlargement of nascent focal adhesions at the ends of the bundle. 3. The transient actomyosin bundles can mature to a cortical stress fiber through recruitment of more actin filaments and NMII.

Article Snippet: LifeAct-TagGFP2 was a kindly provided by the Emmanuel Lemichez lab. Histone H2B-eGFP (in peGFP-N3 backbone) and Histone H2B-mCherry (in pmCherry-N3 backbone) were gifts from Maria Vartiainen lab. miRFP670-paxillin (pPL1514) was constructed by replacing the GFP in eGFP-paxillin (containing gallus gallus paxillin ORF with T132S and M133L unattended mutations in an eGFP-C1 backbone) with miRFP670 originated from miRFP670-N1 (a gift from Vladislav Verkhusha, Addgene plasmid #79987; ) via restriction free cloning using NEBuilder (New England Biolabs).

Techniques: Microscopy, Imaging

Journal: The Journal of Biological Chemistry

Article Title: The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion *

doi: 10.1074/jbc.M114.604876

Figure Lengend Snippet: List of primers Fwd, forward; Ref, reverse.

Article Snippet: The LD ( LEHLLPQL; Ser 640 –Pro 740 ), mutLD ( A EH AA PQI ; Ser 640 –Pro 740 ), and GPIC 714–880 fragments were PCR-amplified from C. caviae GPIC TarP clone ( ) and paxillin LD2 ( LDRLLLEL; Lys 125 –Glu 186 ) from pEGFP-N3-paxillin (Ref. ; obtained from Prof. Rick Horwitz (University of Virginia) through Addgene) using primer pairs 3–4, 5–6, 7–8, and 15–16, respectively.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion *

doi: 10.1074/jbc.M114.604876

Figure Lengend Snippet: Focal adhesion components associate with TarP and its conserved LD-like motif. A , ClustalW sequence alignment showing the putative actin binding domains ( ABD ; open box ) from Tarp orthologs with the conserved residues of the LD motif highlighted in yellow . The red asterisk indicates the recently identified F-actin binding domains (FAB1). The consensus sequence of the LD domain within TarP was aligned with the paxillin LD domain to highlight homology. Species analyzed include C. caviae (GPIC), Chlamydophila abortus ( CAb ), Chlamydophila felis ( CFe ), Chlamydia muridarum ( MoPn ), C. trachomatis serovars A, L2, and D. B , the TarP-LD-like motif was ectopically expressed in Cos7 cells for either 24 or 8 h and tested for the ability to co-recruit pY397-FAK and actin ( white arrowheads ). When transient expression was limited to 8 h rather than 24 h, the TarP-LD specifically localized to FAK-rich focal adhesion structures and to various points along actin stress fibers. Large arrowheads in the 24-h panel indicate the co-recruitment of pY397-FAK and actin to TarP-LD aggregates, whereas in the 8-h panel, they indicate the localization of TarP-LD to focal adhesion structures. Small arrowheads indicate the localization of TarP-LD along the F-actin. Scale bars , 10 μm.

Article Snippet: The LD ( LEHLLPQL; Ser 640 –Pro 740 ), mutLD ( A EH AA PQI ; Ser 640 –Pro 740 ), and GPIC 714–880 fragments were PCR-amplified from C. caviae GPIC TarP clone ( ) and paxillin LD2 ( LDRLLLEL; Lys 125 –Glu 186 ) from pEGFP-N3-paxillin (Ref. ; obtained from Prof. Rick Horwitz (University of Virginia) through Addgene) using primer pairs 3–4, 5–6, 7–8, and 15–16, respectively.

Techniques: Sequencing, Binding Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion *

doi: 10.1074/jbc.M114.604876

Figure Lengend Snippet: Plasma membrane-localized TarP functions to recruit FAK. A and B , DRAQ5-labeled Δ tir EPEC ( red ) bound to cells co-expressing full-length TarP (TirM-TarP-FL; A ) or TirM-Pax-LD2 ( B ) with pEGFP-FAK ( green ) induced a highly localized and transient recruitment event. The images were obtained by live cell microscopy at 30-s intervals. Pixel intensities for representative areas of EGFP-FAK recruitment in TarP-FL- or Pax-LD2-expressing cells were enumerated and plotted as pixel intensity in arbitrary units ( a.u. ) over time. Background fluorescence intensity for neighboring regions was obtained to adjust pixel intensities at the regions of interest. The dotted line represents the average intensity for EGFP-FAK recruitment by Δ tir EPEC in cells expressing the negative control, TirM. Scale bar , 5 μm.

Article Snippet: The LD ( LEHLLPQL; Ser 640 –Pro 740 ), mutLD ( A EH AA PQI ; Ser 640 –Pro 740 ), and GPIC 714–880 fragments were PCR-amplified from C. caviae GPIC TarP clone ( ) and paxillin LD2 ( LDRLLLEL; Lys 125 –Glu 186 ) from pEGFP-N3-paxillin (Ref. ; obtained from Prof. Rick Horwitz (University of Virginia) through Addgene) using primer pairs 3–4, 5–6, 7–8, and 15–16, respectively.

Techniques: Clinical Proteomics, Membrane, Labeling, Expressing, Microscopy, Fluorescence, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion *

doi: 10.1074/jbc.M114.604876

Figure Lengend Snippet: TarP-mediated FAK recruitment is direct and requires the LD domain. A , the clustering ability of the various constructs upon infection by EPEC was evaluated by quantifying pixel intensity and found to be similar. The plasma membrane-clustered labels were specifically monitored because they were the functional subpopulation with regard to signaling and recruitment of FAK and actin. B , diagram of the various TirM-TarP fusion constructs used to identify the region in TarP required for FAK recruitment. The numbers indicate amino acid positions encoded within the C. caviae TarP gene. * denotes full-length TarP. C , adhered EPEC able to recruit phospho-FAK were enumerated for the constructs indicated, and data are represented as a box and whisker plot . Approximately 500–750 particles were counted for each construct. The asterisks indicate significance relative to TirM control (ANOVA; p < 0.005 and post hoc testing Tukey-Kramer; α = 0.01, q = 5.11). D , representative images analyzed to arrive at the data shown in C . Cos7 cells transfected with plasmids encoding progressive TirM-TarP-LD and TirM negative control were infected with Δ tir EPEC to induce clustering of the fusion protein. The white arrowheads indicate colocalization phospho-active FAK (pY397-FAK; green ) with Δ tir EPEC ( blue ). Anti-FAK (phospho-Tyr-397) and anti-HA antibodies were used to visualize pY397-FAK ( green ) or TarP 1–714 /TarP 1–639 /TarP-LD ( red ), respectively. Insets show a magnification of a selected area of the cell. Scale bars , 10 μm. E , the kinetics of EGFP-FAK recruitment was found to be rapid and transient. Pixel intensities for representative areas of EGFP-FAK recruitment to Δ tir EPEC in TarP-LD-expressing cells were enumerated from live cells and plotted as pixel intensity in arbitrary units ( a.u. ) over time. Background fluorescence intensity for neighboring regions was used to normalize the data. Scale bar , 10 μm. E , β-galactosidase activity of E. coli DHM1 strains co-expressing Cya25-FAT and Cya18-TarP-LD, Cya18-Pax-LD2, Cya18-TarP-mutLD, or Cya18-Empty. Activity is expressed as Miller units. ANOVA; p < 0.01 and post hoc testing Tukey-Kramer; α = 0.01, q = 4.91).

Article Snippet: The LD ( LEHLLPQL; Ser 640 –Pro 740 ), mutLD ( A EH AA PQI ; Ser 640 –Pro 740 ), and GPIC 714–880 fragments were PCR-amplified from C. caviae GPIC TarP clone ( ) and paxillin LD2 ( LDRLLLEL; Lys 125 –Glu 186 ) from pEGFP-N3-paxillin (Ref. ; obtained from Prof. Rick Horwitz (University of Virginia) through Addgene) using primer pairs 3–4, 5–6, 7–8, and 15–16, respectively.

Techniques: Construct, Infection, Clinical Proteomics, Membrane, Functional Assay, Whisker Assay, Control, Transfection, Negative Control, Expressing, Fluorescence, Activity Assay