FISIK: Framework for the Inference of In Situ Interaction Kinetics from Single-Molecule Imaging Data.

Recent experimental and computational developments have been pushing the limits of live-cell single-molecule imaging, enabling the monitoring of intermolecular interactions in their native environment with high spatiotemporal resolution. However, interactions are captured only for the labeled subset of molecules, which tends to be a small fraction. As a result, it has remained a challenge to calculate molecular interaction kinetics, in particular association rates, from live-cell single-molecule tracking data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics (FISIK) from single-molecule imaging data with substoichiometric labeling. FISIK consists of (I) devising a mathematical model of molecular movement and interactions, mimicking the biological system and data-acquisition setup, and (II) estimating the unknown model parameters, including molecular association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of the model and data, we adapted the method of indirect inference for model calibration. We validated FISIK using a series of tests in which we simulated trajectories of diffusing molecules that interact with each other, considering a wide range of model parameters, and including resolution limitations, tracking errors, and mismatches between the model and the biological system it mimics. We found that FISIK has the sensitivity to determine association and dissociation rates, with accuracy and precision depending on the labeled fraction of molecules and the extent of molecule tracking errors. For cases where the labeled fraction is too low (e.g., to afford accurate tracking), combining dynamic but sparse single-molecule imaging data with almost-whole population oligomer distribution data improves FISIK's performance. All in all, FISIK is a promising approach for the derivation of molecular interaction kinetics in their native environment from single-molecule imaging data with substoichiometric labeling.

Heterogeneity in VEGF Receptor-2 Mobility and Organization on the Endothelial Cell Surface Leads to Diverse Models of Activation by VEGF.

The dynamic nanoscale organization of cell surface receptors plays an important role in signaling. We determine this organization and its relation to activation of VEGF receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining single-molecule imaging of endogenous VEGFR-2 in live ECs with multiscale computational analysis. We find that surface VEGFR-2 can be mobile or exhibit restricted mobility and be monomeric or non-monomeric, with a complex interplay between the two. This basal heterogeneity results in heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we find that VEGF can bind to monomeric and non-monomeric VEGFR-2 and that, when binding to monomeric VEGFR-2, its effect on dimerization depends on the mobility of VEGFR-2. Our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and the need for multiscale, single-molecule-based analysis to determine its relationship to receptor activation and signaling.

Short-term effects of nCPAP on nasal mucociliary clearance and mucus transportability in healthy subjects.

Nasal mucociliary clearance is a primary defense mechanism of the upper airways and may be acutely affected by nasal continuous positive airway pressure (nCPAP). nCPAP treatment is effective and safe. However, it can cause nasal side effects and contribute to a low compliance to the treatment. The aim of this study was to investigate the short-term effects of nCPAP on nasal mucociliary clearance and on mucus transportability of healthy subjects. Eleven healthy subjects were submitted to 20 min of nCPAP (10 cm H2O). Five subjects were also evaluated before and after 20 min of rest on the consecutive study day. Nasal mucociliary clearance was measured by the saccharin nasal transit time test and nasal mucus was collected for the in vitro study of mucus transportability by the frog palate model, both before and after the nCPAP challenge. Saccharin nasal transit time decreased significantly after nCPAP (9.29+/-6.06 min and 4.83+/-5.57 min; P=0.002 before and after nCPAP respectively). No significant changes were observed on the control day (11.66+/-7.57 min and 12.40+/-5.62 min; P=0.70). Mucus transportability was not significantly affected by nCPAP. Our results suggest that nCPAP can acutely increase nasal mucociliary clearance but does not affect in vitro mucus transportability in healthy subjects.

Sputum induction as a research tool for the study of human respiratory mucus.

The study objectives were to compare in vitro transportability and physical properties of respiratory mucus, obtained invasively by direct collection (DC) right after endotracheal intubation and non-invasively by sputum induction with 3% hypertonic saline solution inhalation (SI) 24 h before the anesthesia. Twenty-two patients with no pulmonary disease scheduled for elective abdominal surgical procedures were studied. The parameters analyzed and the main results are as follows. (1) Transportability by cilia (MCT), SI was higher than DC (0.94+/-0.25 and 0.62+/-0.25; P<0.001). There was a significant correlation between the two methods and DC could be estimated by: DC=0.21+(0.44 SI) (r=0.44; P<0.001). (2) Transportability by cough (CC), SI was higher than DC (68.23+/-32.1 and 33.58+/-19.04 mm; P=0.002). (3) Contact angle (CA), SI was lower than DC (10+/-3 degrees and 22+/-14 degrees ; P=0.025). (4) Rheological properties (no significant difference obtained between SI and DC). These results indicated that SI changes mucus physical properties and transportability in non-expectorators.