Existence of a selective nucleocytoplasmic permeability barrier is attributed to Phenylalanine-Glycine rich proteins (FG-nups) within the central channel of the nuclear pore complex (NPC). Limited understanding of the FG-nup structural arrangement hinders development of strategies directed at disrupting the NPC permeability barrier. In this report we explore an alternative approach to enhancing the NPC permeability for exogenous macromolecules. We demonstrate that the recently discovered inhibitor of clathrin coat assembly Pitstop-2 compromises the NPC permeability barrier in a rapid and effective manner. Treatment with Pitstop-2 causes a collapse of the NPC permeability barrier and a reduction of Importin β binding accompanied by alteration of the NPC ultrastructure. Interestingly, the effects are induced by the same chemical agent that is capable of inhibiting clathrin-mediated endocytosis. To our knowledge, this is the first functional indication of the previously postulated evolutionary relation between clathrin and NPC scaffold proteins.

Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5β1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA-Fn-integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem β-zipper, and that the FnBPA-Fn complex further binds to immobilized α5β1 integrins with a strength much higher than that of the classical Fn-integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites via allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial-host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens.

The vascular endothelium is exposed to three types of mechanical forces: blood flow-mediated shear stress, vessel diameter-dependent wall tension and hydrostatic pressure. Despite considerable variations of blood pressure during normal and pathological physiology, little is known about the acute molecular and cellular effects of hydrostatic pressure on endothelial cells. Here, we used a combination of quantitative fluorescence microscopy, atomic force microscopy and molecular perturbations to characterize the specific response of endothelial cells to application of pressure. We identified a two-phase response of endothelial cells with an initial response to acute (1 h) application of pressure (100 mmHg) followed by a different response to chronic (24 h) application. While both regimes induce cortical stiffening, the acute response is linked to Ca-mediated myosin activation, whereas the chronic cell response is dominated by increased cortical actin density and a loss in endothelial barrier function. GsMTx-4 and amiloride inhibit the acute pressure response, which suggests that the ENaC Na channel is a key player in endothelial pressure sensing. The described two-phase pressure response may participate in the differential effects of transient changes in blood pressure and hypertension.

The collagen-binding protein Cna is a prototype cell surface protein from Staphylococcus aureus which fulfils important physiological functions during pathogenesis. While it is established that Cna binds to collagen (Cn) via the high-affinity collagen hug mechanism, whether this protein is engaged in other ligand-binding mechanisms is poorly understood. Here, we use atomic force microscopy to demonstrate that Cna mediates attachment to two structurally and functionally different host proteins, i.e., the complement system protein C1q and the extracellular matrix protein laminin (Lam), through binding mechanisms that differ from the collagen hug. We show that single Cna-C1q and Cna-Lam bonds are much weaker than the high-affinity Cna-Cn bond and that their formation does not require the B-region of Cna. At the whole cell level, we find that bacterial adhesion to C1q-substrates involves only one (or two) molecular bond(s), while adhesion to Lam is mediated by multiple bonds, thus suggesting that multivalent or cooperative interactions may enhance the strength of adhesion. Both C1q and Lam interactions can be efficiently blocked by monoclonal antibodies directed against the minimal Cn-binding domain of Cna. These results show that Cna is a multifunctional protein capable of binding to multiple host ligands through mechanisms that differ from the classical collagen hug.