PMC

LA treatment disrupts in a two-step process the two actin networks in bridges. (A, B) LA treatment (applied at t=0 min) disrupts bridges by detachment of the bow-tie actin network from the borders of the FN stripes (white arrowheads). (C) LA treatment (applied at t=0) showed two distinct steps: phase 1-actin network detachment from the FN stripes with rapid actin fibres condensation (red, grey and white arrows) followed by phase 2-slow bridge retraction (arrowhead) during which the remaining diffuse actin meshwork contracts and displays actin asters formation and interaction (box i). Scale bars: (A, B) 30 μm and (C) 15 μm.

Formation and extension of bridges correlate with actomyosin assembly and contractions. (A) Differential interference contrast: formation of arch-shaped edges (arrowheads) during late spreading on a patterned substrate. Arrows indicate coexisting transient protrusions. GFP-β-actin (TIRF): when bridges formed (2.8 min), actin cables bent towards the middle (box ii, arrowheads, 7.0 min). GFP-MRLC (TIRF): when bridges formed, MRLC is enriched above non-adhesive regions and condensed into bow-tie-shaped fibres at longer times. (B) Bridge extension during a cycle of extension of actin-rich protrusions (arrow) followed by contraction into MII bundles (arrowhead). Plot of positions of FN-attached regions (blue and red dots) and bridge edge (green dot) versus time during one cycle. Scale bars: (A) 15 μm (except for differential interference contrast panel: 18 μm) and (B) 13 μm.

Actomyosin dynamics within bridges exhibit two distinct patterns, (1) ‘contractile' treadmilling at the concave edges of bridges and (2) ‘cross-linking' in the body of bridges. (A) GFP-α-actinin (TIRF): speckles decorated the ‘bow-tie'-shaped actin cytoskeleton as well as FAs; velocity field: particle image velocimetry analysis of speckles' dynamics permitted to define a ‘contractile' treadmilling pattern (region 1-red arrows) at concave edges of mature bridges and in newly formed bridges, and a ‘cross-linking' pattern (region 2-blue arrows) in the body of bridges. Velocity scale bar=25 nm/s. (B) GFP-MRLC (TIRF): similar dynamical patterns were observed with MII ongoing (i) ‘contractile' treadmilling in region 1 as seen with MRLC speckles in snapshot (arrowhead) and in kymograph taken along the red line (arrowhead: corresponding speckle trajectory) and (ii) ‘cross-linking' dynamics in region 2 as seen in kymograph of MRLC speckles along actin fibre (blue line ii). Images were filtered with the ImageJ plugin SpotTracker. Scale bars: (A, B) 15 μm.

Working model of the maintenance and translocation of cellular bridges controlled by actomyosin contraction. Bridges between the FN stripes have a ‘bow-tie'-shaped actin cytoskeleton. Such organization is maintained in regions 1 by MIIA-based continuous actomyosin treadmilling pattern located at the concave edges (contraction nodes are represented with yellow ovals) and in region 2 by a ‘cross-linking' pattern in the body of bridges. Extensions on FN stripes provide new FA sites, which serve as anchors for bridges' actomyosin meshwork to translocate using contraction-generated forces. The sequence of events in regions 1 are (1) generation of forces (yellow arrows) on FN stripes, which induce (2) maturation of adhesions on edges of FN stripes (green rectangle), (3) forces localize at FAs sites, the turnover of actin (blue chevron) and subsequent MII (red circles) association, which maintain the (4) contractile treadmilling flow (red arrow) of actin and MII from FN stripes towards non-adhesive area. In the region 2, actomyosin bundles are pulled on both sides by contractions in regions 1 (yellow arrows). This induces (1) tension in actomyosin bundles observed through (2) oscillating ‘cross-linking' pattern of MII (orange circles) and α-actinin. When tension is too high, cross-links ruptured and bundles are tearing until (3) MII-based healing process during which new MIIB cross-linkers associate and restore the mechanical continuity. Such process confers tensional homeostasis to the dynamic meshwork.

Generation of forces induced by MII activity is essential for the formation and maintenance of bridges. (A) Reversible collapse of bridges induced by MII inhibition with MEF initial spreading (t=0 to 40 min), perfusion of BBI (t=50 to 100 min), and drug washout (t=100 min). (B) Plot of the variation versus time of the total projected area, A_TOTAL (black circles), the bridge area, A_BRIDGE (red circles) and the cell area above the adhesive region, A_FN (green circles), for the cell depicted in (A). (C) Plot of the variation versus time of the bridging index, Br_i (black circles), for the cell depicted in (A). Br_i was constant during cell spreading and decreased from 69 to 32% during BBI perfusion. (D) During BBI treatment, force generation by bridges represented with red arrows (force scale bar=5 nN) stopped after 5–10 min (red circles indicated pillars where force generation was stopped); then bridge tearing (left panel: arrowheads) and/or bridge retraction (middle panel) led to bridges' collapse. Plot of the variation during BBI perfusion versus time of the traction force exerted on pillars adjacent to the concave edges (marked by asterisks: yellow for pillar in left panel and blue in middle panel). Scale bars: (A) 30 μm and (D) 15 μm.

MII activity increased actin filament assembly at the adhesion sites and cross-linking of actomyosin filaments in the body of bridges. (A) GFP-β-actin (TIRF): under BBI treatment, actin cables within the bridge vanished concomitantly with the decrease of A_BRIDGE and with further expansion on FN stripes. (B) GFP-α-actinin (TIRF) speckles dynamics in the actomyosin bundles in region 2 during BBI treatment as seen in kymograph (box i) taken along actomyosin fibres (blue line i). (C) FRAP experiment with GFP-β-actin. Left: GFP-β-actin in region 1 was photo-bleached in a square of 12 μm side (white square with dashed lines) in the same cell for two different conditions: left panel represents the control case; right panel shows the case of the same cell treated with BBI. Dashed white segments outlined in the control case the new position of the photo-bleached actin bundles that moved away from their anchoring point. Right: montage of the actin bundles inside the red rectangles (15.2 × 1.8 μm) for the two conditions. Scale bars: (A) 18 μm, (B) 15 μm and (C) 8.5 μm.

MII isoforms localized differentially in bridges with MIIB in the body of bridges and MIIA at the concave edges. (A) GFP-MRLC (TIRF): MII inhibition arrested healing of broken fibres in region 2 and stopped the inwards contractile treadmill of MRLC speckles in region 1. (i) kymograph of MRLC speckles along a breaking actin fibre (blue line i) before and during BBI treatment and (ii) kymograph of MRLC speckles along actin fibre (red line ii) before and during BBI treatment. (B) Top panel: MIIA localized at the vicinity of concave edges (region 1) as seen in GFP-MIIA transfected MEFs by TIRF (left) or in MEFs immunostained against MIIA by EPI (right). Bottom panel: MIIB localized in the body of bridges (region 2) as seen in GFP-MIIB transfected MEFs by TIRF (left) or in MEFs immunostained against MIIB by EPI (right). (C) Differential interference contrast micrograph of control MEFs and MIIA knockdown MEFs (detected by GFP expression as seen in EPI image in inset) 25 min after plating. Plot of the mean bridging index Br_i versus time during spreading for control MEFs (open circles) and MIIA-depleted cells (closed circles) in differential interference contrast micrograph. Histogram representing the percentage of cells after 30 min on FN stripes, which are displaying bridges (white), spread without bridges (black), and unspread (light grey) for respectively, MEFs treated with Mock siRNA and MIIA siRNA. Scale bars: (A, B) 15 μm and (C) 30 μm.

Cellular bridges are actin-rich structures characterized by ‘bow-tie'-shaped cytoskeleton and concave edges. FA and strongest traction forces are generated at the sides of concave edges. (A) Typical morphology of MEFs spread for 2 h on patterned FN lines. (i) Close-up of cell (i) in the outlined box (Br_i=66%). Top panel: bridges highlighted in red (A_BRIDGE) developed above the non-adhesive regions with arch-shaped ends. The cell regions in contact with FN lines are in green (A_FN); bottom panel: actin cytoskeleton in the same cells (EPI and TIRF images). (B) Adhesion proteins: paxillin immunostaining by EPI and with GFP-integrin β3 by TIRF. (C) Top left: original geometry of PDMS force-sensing pillar device; bottom left: scanning electron micrograph of MEFs onto pillars; top right: differential interference contrast micrograph of MEF exerting onto pillars, traction forces represented with red arrows (force scale bar=12 nN); bottom right: plot of the amplitude (red circles) of forces exerted on each pillars of the bottom row in the differential interference contrast micrograph, as well as separated force components: Fx along the axis of the rows (green circles) and Fy directed perpendicularly (blue circles). FN lines are outlined with yellow dashed lines. Scale bars: (A) 50 μm, A (i) 30 μm, (B, C) 15 μm.