PIP2-induced membrane binding of the Vinculin tail competes with its other binding partners.

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its Tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-Vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, where unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond long all-atom simulations. This allowed us to map the interaction of the Vinculin Tail with PIP2-enriched membranes at atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between Vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the Basic Collar and the Basic Ladder, as the main PIP2 interaction sites. By introducing a valency disaggregated binding network analysis, we were able to map the protein lipid interactions at unprecedented detail. In contrast to the Basic Collar where PIP2 is specifically recognized by an up to hexavalent binding pocket, the Basic Ladder forms a series of low valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining Vinculin in a closed, auto-inhibited conformation. These findings led us to propose a molecular mechanism for the coupling between Vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-Actin binding that disfavors simultaneous interaction with both ligands despite non-overlapping binding sites

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

The effects of the net advancement speed of the cell edge on the contact time between the lamellipodium and a filopodia adhesion.

<p><b>A.</b> Scatter plot of the part of the contact time of a filopodia adhesion with the advancing lamellipodium, with respect to the net advancement speed of the cell edge. All data were for REF52-β3-integrin-EGFP cells spreading on FN coated glass. <i>Black:</i> maturing filopodia adhesions. <i>Red:</i> disassembling filopodia adhesions. Due to the fast net advancement speed of the cell edge, the disassembling filopodia adhesions demonstrated a reduced contact time with the advancing lamellipodia, which subsequently had very brief of no pausing at these filopodia adhesions. Open symbols correspond to the filopodia adhesions in B–b. <b>B. a.</b> A fast spreading REF52 fibroblast expressing β3-integrin-EGFP (green) on FN coated glass (5 min–9 min 30 s after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s013" target="_blank">Video S8</a>). The cell membrane was stained with the fluorophore DiI (red). <b>b.</b> Magnified views of the dashed rectangle regions (I and II) in a. The selected frames from the time-lapse sequences showed the disassembly (I, horizontal arrows) or steady maturation (II, vertical arrows) of the filopodia adhesions as a result of ongoing advancement or prolonged pausing of lamellipodium at the two filopodia adhesions respectively. <b>c.</b> The advancement of cell two edges are represented by the kymographs that were generated along the corresponding kymograph lines (colored arrows in a) from the membrane fluorescence signal at the two filopodia adhesions. The dashed lines indicate the advancing (yellow) and pausing (pink) phases of lamellipodia at the respective filopodia adhesions. The net advancement speeds of the cell edges were measured at the corresponding sections of the kymographs as indicated by the dashed yellow lines (B-c I, 161 nm/s; B–c II, 147 nm/s). Scale bars: 2 µm (B–b), 5 µm (B–a).</p

The actin cytoskeleton organization at filopodia adhesions as seen in ventral cell membrane samples.

<p>Cells were gently blasted open. While this might cause some local disruptions, the main purpose was to distinguish between molecular components that are weak versus strongly bound to the ventral site of the plasma membrane. The colors of stains denote the ventral cell membrane (red) and actin structures (green). Filopodia adhesions were identified (see method) by vinculin immunostaining (cyan, A–b, B–b), interference reflection signals (grey images, B), and β3-integrin-EGFP imaging (grey, C). <b>A and B.</b> Cell edge regions of the exposed ventral cell membranes from HFF cells (20 min (A) or 33 min (B) after plating on FN coated glass). The white square regions (A–a, B–a) were magnified respectively (b, c in A; b–f in B). In merged images, vinculin was alternatively false colored in red (A–c, B–e, B–f) for a better presentation of the colocalization of signals. Pink arrow or arrowheads denote the former filopodia actin bundles and their associated substrate adhesions. The cyan arrowheads indicate circumferential stress fibers with their surface anchorage (A) or tight surface association (B). The white arrow (A–c) indicates the separation between the filopodia adhesion in the cell lamellum and the distal section of the filopodium, which might be fracture that could have occurred during the sample preparation. <b>C.</b> The cell periphery of an exposed ventral cell membrane from a REF52-β3-integrin-EGFP cell (30 min after plating on FN coated glass) showed a β3-integrin cluster at the filopodium (pink arrows), circumferential stress fibers (cyan arrowheads) anchored to the substrate via the β3-integrin clusters formed at the side of this filopodia adhesion, and the sidewise widening of the filopodia β3-integrin cluster (pink arrowheads) following the connected thick circumferential stress fibers in the cell lamellum. Scale bar: 5 µm (A–b & c, B, C), 10 µm (A–a).</p

Engineering Mechanosensitive Multivalent Receptor–Ligand Interactions: Why the Nanolinker Regions of Bacterial Adhesins Matter

Inspired by bacterial adhesins, we present a promising strategy of how to engineer peptides to probe various mechanical strains of extracellular matrix fibers. Functional sequence alignment of bacterial adhesins reveals that the bacterial linkers connecting the multivalent binding motifs recognizing fibronectin show considerable heterogeneity in length. Their length regulates the tunable affinities for fibronectin fibrils when stretched into different mechanical strain states. This platform has potential applications in probing extracellular matrix fiber strains in tissues

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin‑7

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (₁₁₀TKSLEEN₁₁₆ and the truncated ₁₁₂SLEE₁₁₅ in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI₂) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies

Cycles of periodic protrusions and retractions of a lamellipodium in the proximity of maturing filopodia adhesions.

<p><b>A.</b> The β3-integrin-EGFP (green) expressing REF52 fibroblast on a FN coated glass surface was time-lapse tracked with confocal microscopy. The cell membrane was stained by the fluorophore DiI (red). <b>B.</b> Magnified views of the white rectangle region in A showed the filopodia adhesion at the start (left) and end (right) of the time-lapse tracking sequence (20 min–47 min after seeding, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s008" target="_blank">Video S3</a>). <b>C.</b> Time-lapse montages of the white rectangle region in A. At the time point indicated by the grey asterisk, we had to increase the laser (488 nm) intensity to compensate for photobleaching. To better visualize lamellipodium activities, the presented montage was stretched vertically 2×. <b>D.</b> Histograms of the distances, durations and speeds of the cyclic protrusions (black bars) and retractions (brown bars) of lamellipodia in proximity to filopodia adhesions. Values in the parenthesis gave the number of measurements (at 14 filopodia adhesions in 5 cells). Scale bars: 1 µm (B), 10 µm (A). The horizontal grey bar indicates the width of a single image frame in the montage in C (2.5 µm).</p

Growth kinetics of filopodia adhesions after MLCK inhibition (A, B) or on soft polyacrylamide gels (C, D).

<p>The β3-integrin-EGFP (green) expressing REF52 fibroblasts on FN coated glass (A, B) or polyacrylamide gel (C, D) surfaces were time-lapse tracked with confocal microscopy. The cell membrane was stained by the fluorophore DiI (red). <b>A and B.</b> Inhibition of MLCK by ML-7 suppressed the cyclic protrusions and retractions of lamellipodium and the growth of filopodia adhesions. <b>A.</b> Time-lapse montage (b) of the filopodium containing cell edge region (white rectangle, a) showed the loss of the cyclic protrusions and retractions of the lamellipodium in the proximity of the filopodium in ML-7 treated cells (25 min–30 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s009" target="_blank">Video S4</a>). <b>B.</b> Time-lapse montage (b) of the filopodia adhesion cell edge region (white rectangle, a) demonstrated the suppressed size growth of the filopodia β3-integrin adhesion in ML-7 treated cells. c. Comparison of the area (average of the tracked temporal states (0–600 s)) of the filopodia β3-integrin adhesions (7 adhesions, n = 17) in the ML-7 (30 or 40 µM) treated cells with that of the maturing filopodia β3-integrin adhesions (6 adhesions, n = 164) in the cells plated in normal culture media. Error bars correspond to the standard errors. <b>C and D.</b> The growth of filopodia adhesions in REF52-β3-integrin-EGFP cells on soft PAA substrates was associated with the restored cyclic protrusions and retractions of lamellipodia. The PAA substrate (7.4 kPa) was covalently coated with FN. <b>C.</b> Cell without stable surface adhesions (a, 103 min–113 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s010" target="_blank">Video S5</a>) exhibited significant ruffling as shown by the time-lapse montage of the cell edge (b). <b>D.</b> The filopodia adhesion (white rectangle, b) at the edge of a cell (white square in a, 121 min–136 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s011" target="_blank">Video S6</a>) matured in association with the restored cyclic protrusions and retractions of the lamellipodium (c). To better visualize lamellipodium activities, all montages were stretched vertically 2×. Scale bars: 5 µm (A, D–b), 10 µm (B, C, D–a). The width of a single image frame in the montage is indicated by the horizontal grey bar: A–b, 2.5 µm; B–b, 4.5 µm; C–b, 6.2 µm; D–c, 3 µm.</p

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin‑7

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (₁₁₀TKSLEEN₁₁₆ and the truncated ₁₁₂SLEE₁₁₅ in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI₂) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies