Direct quantification of biomolecular interaction by single-molecule force spectroscopy has evolved into a powerful tool for materials and life sciences. We introduce an approach in which the unbinding forces required to break intermolecular bonds are measured in a differential format by comparison with a known reference bond (here, a short DNAduplex). In addition to a marked increase in sensitivity and force resolution, which enabled us to resolve single–base pair mismatches, this concept allows for highly specific parallel assays. This option was exploited to overcome cross-reactions of antibodies in a protein biochip application.

Together with droplet creation, fusion and sorting, the incubation of droplets is one of the most important and essential operations for droplet-based microfluidic assays. This manuscript concerns the development of delay-lines, which are necessary to allow incubation of reactions for precise time periods. We analyze the problems associated with creating delay-lines for incubation in the minute to hour time range, which arise from back-pressure and from the dispersion in the incubation time due to the unequal speeds with which droplets pass through the delay-line. We describe delay-line systems which resolve these problems and demonstrate their use to measure reaction kinetics over several minutes in droplets.

In the cell, enzymes are almost always spatially confined in crowded and tightly controlled cellular compartments. The entrapment of enzymes in artificial nanoreactors as biomimetic systems can be expected to contribute to the understanding of the activity and the interactions of enzymes in confined spaces. The capsid of the Cowpea Chlorotic Mottle virus (CCMV) represents such an artificial nanoreactor that can be used to encapsulate multiple proteins in its interior. Employing a controlled encapsulation process we are able to load a precise number of proteins (Pseudozyma antarctica lipase B and EGFP) into the CCMV capsid and to study their activity. In the case of the enzyme, our results indicate that the apparent overall reaction rate increases upon encapsulation and is almost independent of the number of enzymes in the capsid. These observation are the result of the extremely high confinement molarity of the enzyme inside the capsid (M_conf = ∼ 1 mM) leading to very rapid formation of the enzyme–substrate complex. These results highlight the importance of small volumes for efficient multi-enzyme cascade catalysis.

Balanced transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP)-signaling is essential for tissue formation and homeostasis. While gain in TGFβ signaling is often found in diseases, the underlying cellular mechanisms remain poorly defined. Here we show that the receptor BMP type 2 (BMPR2) serves as a central gatekeeper of this balance, highlighted by its deregulation in diseases such as pulmonary arterial hypertension (PAH). We show that BMPR2 deficiency in endothelial cells (ECs) does not abolish pan-BMP-SMAD1/5 responses but instead favors the formation of mixed-heteromeric receptor complexes comprising BMPR1/TGFβR1/TGFβR2 that enable enhanced cellular responses toward TGFβ. These include canonical TGFβ-SMAD2/3 and lateral TGFβ-SMAD1/5 signaling as well as formation of mixed SMAD complexes. Moreover, BMPR2-deficient cells express genes indicative of altered biophysical properties, including up-regulation of extracellular matrix (ECM) proteins such as fibrillin-1 (FBN1) and of integrins. As such, we identified accumulation of ectopic FBN1 fibers remodeled with fibronectin (FN) in junctions of BMPR2-deficient ECs. Ectopic FBN1 deposits were also found in proximity to contractile intimal cells in pulmonary artery lesions of BMPR2-deficient heritable PAH (HPAH) patients. In BMPR2-deficient cells, we show that ectopic FBN1 is accompanied by active β1-integrin highly abundant in integrin-linked kinase (ILK) mechano-complexes at cell junctions. Increased integrin-dependent adhesion, spreading, and actomyosin-dependent contractility facilitates the retrieval of active TGFβ from its latent fibrillin-bound depots. We propose that loss of BMPR2 favors endothelial-to-mesenchymal transition (EndMT) allowing cells of myo-fibroblastic character to create a vicious feed-forward process leading to hyperactivated TGFβ signaling. In summary, our findings highlight a crucial role for BMPR2 as a gatekeeper of endothelial homeostasis protecting cells from increased TGFβ responses and integrin-mediated mechano-transduction.