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.

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-ß expression. Enhanced IFN-ß expression subsequently increases pro-inflammatory gene expression via the IFN-ß-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-ß-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.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

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 αIIbß3 rather than α5ß1 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.