Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals

Abstract

How cells sense their mechanical environment and transduce forces into biochemical signals is a crucial yet unresolved question in mechanobiology. Platelets use receptor glycoprotein Ib (GPIb), specifically its α subunit (GPIbα), to signal as they tether and translocate on von Willebrand factor (VWF) of injured arterial surfaces against blood flow. Force elicits catch bonds to slow VWF–GPIbα dissociation and unfolds the GPIbα leucine-rich repeat domain (LRRD) and juxtamembrane mechanosensitive domain (MSD). How these mechanical processes trigger biochemical signals remains unknown. Here we analyze these extracellular events and the resulting intracellular Ca2+ on a single platelet in real time, revealing that LRRD unfolding intensifies Ca2+ signal whereas MSD unfolding affects the type of Ca2+ signal. Therefore, LRRD and MSD are analog and digital force transducers, respectively. The >30 nm macroglycopeptide separating the two domains transmits force on the VWF–GPIbα bond (whose lifetime is prolonged by LRRD unfolding) to the MSD to enhance its unfolding, resulting in unfolding cooperativity at an optimal force. These elements may provide design principles for a generic mechanosensory protein machine.DOI: http://dx.doi.org/10.7554/eLife.15447.001

Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis of Signal Crosstalk between Multiple Molecular Species

“…3I). This is consistent with the previous observation that α-type Ca 2+ serves as a platelet activating signal 24 and indicates that GPIIb-IIIa upregulation is sustained for minute time elapse after GPIb triggered calcium spike has returned to the basal level. Our results are also consistent with the widely accepted model that GPIIb-IIIa assumes a low-affinity state on resting platelets 35 .…”

“…3F-H). In the 200-s observation time window, three types of intraplatelet Ca 2+ signals were observed as previously characterized 24 . These are: 1) null-type Ca 2+ , featured by a basal trace with a minimum intensity increase (Fig.…”

“…Biotinylated recombinant pMHC monomers OVA:H2-K b and OVA:H2-K b α3A2 were from the National Institutes of Health Tetramer Core Facility at Emory University. Recombinant VWF-A1 monomeric domain (residues 1238-1471) was a kind gift from Miguel Cruz (Baylor College of Medicine, Houston, TX) as described previously 24,39,46 . Human fibronectin (cat # 33016015; ThermoFisher Scientific), anti-CD62p-FITC antibody (AK4; BD Bioscience), calcium indicator Fura2-AM (Life Technologies) were purchased.…”

“…Interestingly, the formation of the (TCR/CD8)-pMHC trimolecular bond exhibits a time delay and requires intracellular signaling via Src family kinases [18][19][20] whereas that of the (α 5 β 1 /Sydecan-4)-Thy-1 bond is elicited by pulling forces 21 . The mechanically based methods have also been combined with fluorescence imaging to enable concurrent observations of receptor-ligand binding and intracellular signaling so induced, to allow for correlative analysis over time at the single-cell level [22][23][24] . A major limitation of the existing mechanically based methods is the inability to control the separate engagement of multiple receptor-ligand species.…”

“…3D,E). To observe intraplatelet Ca 2+ signals triggered by GPIb mechanoreception, the platelets were preloaded with a fluorescent dye (Fura-2 AM) and the simultaneous Ca 2+ ratiometric imaging optics were integrated as previously described 13,24,30 . To separate engagements of the two receptors in time, we first drove the platelet to repeatedly touch Probe 1 coated with A1 (or BSA as a control) (Fig.…”