Platelets drive fibronectin fibrillogenesis using integrin αIIbβ3

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair

Attending [to] Futures : Matters of Politics in Design Education, Research, Practice

Acknowledging the ways in which design (as practices, forms of knowledge, and sets of objects) is accountable for ongoing social and environmental injustices, this anthology contains contributions that envision alternative ways of exploring and designing more livable futures. Attending to these futures requires a reckoning with a multiplicity of actors and contexts, from institutional norms and regulations, to pedagogies, curricula, programs, digital tools, infrastructures, and architectural environments. Last but not least, attention is drawn to the mechanisms and protocols by which these futures are imagined and shaped. This includes critically examining the ways in which design is talked about, taught, and learned in order to empower future designers to engage with the political issues, cultural conditions, and social and environmental implications of their work

Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

Mutations and defects in nuclear lamins can cause major pathologies, including inflammation and inflammatory diseases. Yet, the underlying molecular mechanisms are not known. We now report that the pro-inflammatory activation of macrophages, as induced by LPS or pathogenic E. coli, reduces Lamin-A/C levels thereby augmenting pro-inflammatory gene expression and cytokine secretion. We show that the activation of bone-marrow-derived macrophages (BMDMs) causes the phosphorylation and degradation of Lamin-A/C, as mediated by CDK1 and Caspase-6, respectively, necessary for upregulating IFN-β expression. Enhanced IFN-β expression subsequently increases pro-inflammatory gene expression via the IFN-β-STAT axis. Pro-inflammatory gene expression was also amplified in the complete absence of Lamin-A/C. Alternatively, pharmacological inhibition of either Lamin-A/C phosphorylation or degradation significantly downregulated pro-inflammatory gene expression, as did the targeting of IFN-β-STAT pathway members, i.e. phospho-STAT1 and phospho-STAT3. As Lamin-A/C is a previously unappreciated regulator of the pro-inflammatory macrophage response, our findings suggest novel opportunities to treat inflammatory diseases.ISSN:2589-004

Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response

Mutations and defects in nuclear lamins can cause major pathologies, including inflammation and inflammatory diseases. Yet, the underlying molecular mechanisms are not known. We now report that the pro-inflammatory activation of macrophages, as induced by LPS or pathogenic E. coli, reduces Lamin-A/C levels thereby augmenting pro-inflammatory gene expression and cytokine secretion. We show that the activation of bone-marrow-derived macrophages (BMDMs) causes the phosphorylation and degradation of Lamin-A/C, as mediated by CDK1 and Caspase-6, respectively, necessary for upregulating IFN-β expression. Enhanced IFN-β expression subsequently increases pro-inflammatory gene expression via the IFN-β-STAT axis. Pro-inflammatory gene expression was also amplified in the complete absence of Lamin-A/C. Alternatively, pharmacological inhibition of either Lamin-A/C phosphorylation or degradation significantly downregulated pro-inflammatory gene expression, as did the targeting of IFN-β-STAT pathway members, i.e. phospho-STAT1 and phospho-STAT3. As Lamin-A/C is a previously unappreciated regulator of the pro-inflammatory macrophage response, our findings suggest novel opportunities to treat inflammatory diseases

Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes

The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis

Clathrin coats partially preassemble and subsequently bend during endocytosis

Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargo. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle. Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area before they become rapidly and continuously bent, and this mechanism is confirmed in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.ISSN:0021-9525ISSN:1540-814

Platelets drive fibronectin fibrillogenesis using integrin αIIbβ3

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.ISSN:2375-254

Platelets exploit fibrillar adhesions to assemble fibronectin matrix revealing new force-regulated thrombus remodeling mechanisms

Upon vascular injury, platelets are crucial for thrombus formation and contraction, but do they directly initiate early tissue repair processes? Using 3D super-resolution microscopy, micropost traction force microscopy, and specific integrin or myosin IIa inhibitors, we discovered here that platelets form fibrillar adhesions. They assemble fibronectin nanofibrils using αIIbβ3 (CD41/CD61, GPIIb-IIIa) rather than α5β1 integrins, in contrast to fibroblasts. Highly contractile platelets in contact with thrombus proteins (fibronectin, fibrin) pull fibronectin fibrils along their apical membrane, whereas platelets on basement membrane proteins (collagen IV, laminin) are less contractile generating less stretched planar meshworks beneath themselves. As probed by vinculin-decorated talin unfolding, platelets on fibronectin generate similar traction forces in apical fibrillar adhesions as fibroblasts do. These are novel mechanobiology mechanisms by which platelets spearhead the fibrillogenesis of the first de novo ECM, including its 2D versus 3D network architectures depending on their ECM environment, and thereby pave the way for cell infiltration