Elastic Coupling of Nascent apCAM Adhesions to Flowing Actin Networks

<div><p>Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.</p></div

Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling

Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(−/−)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(−/−) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis

Membrane deforms but is too weak to explain observed traction forces.

<p>(A) Schematic representation of the setup: a 2 µm diameter apCAM-coated bead with two 100 nm fluorescent beads irreversibly attached on its surface is placed on the membrane of growth cone treated with 5 µM latrunculin B. (B) Displacement over time of the apCAM-coated bead (green) and the two fluorescent beads (red and blue) along the x-axis. (C) Positions in the x-y plane of the two fluorescent beads in the frame of reference of the center of mass of the apCAM-coated bead (green circle indicates the bead’s circumference). (D) Translational mean squared displacement of the apCAM-coated bead (green) and rotational mean squared displacements of the twisting angle (red) and rolling angle (blue). (E) Schematic representation of the setup: a 2 µm diameter apCAM-coated bead with a 100 nm diameter fluorescent bead irreversibly attached on its surface is placed on the membrane of a control growth cone. (F) Displacement over time of the apCAM-coated bead (green) and the small fluorescent bead (orange) along the actin flow. Green force scale bar applies only to the green trace. Rolling angle (blue) calculated from the small bead motion respect to the apCAM-coated bead motion. (G) Breakage force histograms calculated from centroids of the apCAM-coated bead (blue) and the fluorescent beads on its surface (red).</p

Schematic mechanical model of a nascent adhesion.

<p>The trapped bead alternates between frictional coupling with retrograde flow (left) and intermittent coupling to elastic intracellular structure (rignt).</p

Elastic properties of nascent adhesions.

<p>(<i>A</i>) Top panel: Bead (green) vs. trap (red) relative positions. Flow-coupled apCAM-coated bead (green) is successively clamped at different constant forces by changing the separation between the bead and the center of the optical trap (red). Middle panel: flow-coupled bead is first released from the optical trap (“unload” arrow) and then force-clamped (“reload” arrow) at 30, 20 and 10 pN successively. Bottom panel: bead velocity exhibits a transient for each change of force indicated by the black arrows (B) Steady bead velocity under constant force normalized to bead velocity under zero force. Same color symbols correspond to same bead clamped at different forces. For each particular experiment the laser power of the trap is first set and then the distance between trap and bead was adjusted. (C) Jump sizes of flow-coupled beads when released from (Δ<i>x</i>_{<i>unload</i>}) and reloaded by(Δ<i>x</i>_{<i>reload</i>}) optical traps. Same color symbols correspond to different events with the same bead and cell. (D-E) When a flow-coupled bead (green) was displaced by 0.7 µm from the trap center (red), the bead was released. Dashed lines in D and E depict the extrapolated position of actin features moving with retrograde flow. (F) In-plane force-displacement curve of nascent adhesion. Displacement is defined as the distance between the bead and the blue dashed line in D representing the trajectory of an unrestrained flow-coupled bead. (G) Force-velocity of the flow-coupled bead during relaxation calculated using bead trajectory in <i>F</i> and force-displacement curve in <i>H</i>.</p

Relaxation of bead-substrates released from optical tweezers.

<p>(<i>A</i>) <b>apCAM-coated bead (green) is restrained 10 s in a stationary trap (red)</b>. </p> <p>The trap is then turned off and the bead moves with actin retrograde flow. A closed-loop trapping system returns the bead to its original position after it has been displaced by about 3 µm. Shaded and white areas represent periods where the optical tweezers are on and off, respectively. An enlargement showing bead displacement kinetics before and after release (boxed region) is shown below. (<i>B</i>) Histogram of transient displacements defined as the distance traveled by the bead during the first 0.8 s after turning laser off. Inset: histogram of the steady state bead velocity after each transient event: 0.8 s after release, the steady-state velocity was measured over the next 8 to 12 s. (<i>C</i>) Transient displacements versus force applied to beads just before turning off the laser. Note that 100% of beads exhibited elastic relaxation when force was >10 pN. (<i>D</i>) Histogram of transient displacement for a growth cone treated with 50 µM blebbistatin. Inset: histogram of the steady state bead velocity after each transient. (<i>E</i>) Bar graph showing the percentage of beads exhibiting no jump (blue), and the percentage exhibiting a jump (red), in control or blebbistatin-treated experiments. The first and third plots include all events after laser turn-off, while the second and fourth plots include events where the pre-stress exceeded 10 pN.</p

Correlation of bead movements with actin flow.

<p>(A) Schematic depicting growth cone cytoplasmic domains and an apCAM coated bead restrained against retrograde flow on the surface of an <i>Aplysia</i> bag cell growth cone using an optical trap. (B) DIC image of trapped apCAM-coated bead. (C) F-actin labeled with fluorescent phalloidin imaged with confocal microscopy. (D) Kymograph of actin speckle displacement sampled along the yellow line in (C). Green line shows trapped bead trajectory. Magenta lines show F-actin retrograde flow speed. Red arrows denote breakage events. The yellow arrow indicates the point of maximum optical force where the bead escapes from the trap. (E) Time-lapse montage DIC (top) and phalloidin-tagged actin (bottom) of a bead forming an adhesion, coupling to retrograde flow, a breakage event followed by recoupling to actin flow and eventual bead escape. Bead positions are superimposed on the phalloidin montage (green circles). Red arrow: breakage event; white/black arrow, bead escape. Time and space for C-D indicated by respective scale bars.</p