Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

The patch-clamp method, which was awarded the Nobel Prize in 1991, is a well-established and indispensable method to study ion channels in living cells and to biophysically characterize non-voltage-gated ion channels, which comprise about 70% of all ion channels in the human genome. To investigate the biophysical properties of non-voltage-gated ion channels, whole-cell measurements with application of continuous voltage ramps are routinely conducted to obtain current-voltage (IV) relationships. However, adequate tools for detailed and quantitative analysis of IV curves are still missing. We use the example of the transient receptor potential classical (TRPC) channel family to elucidate whether the normalized slope conductance (NSC) is an appropriate tool for reliable discrimination of the IV curves of diverse TRPC channels that differ in their individual curve progression. We provide a robust calculation method for the NSC, and, by applying this method, we find that TRPC channel activators and modulators can evoke different NSC progressions independent from their expression levels, which points to distinguishable active channel states. TRPC6 mutations in patients with focal segmental glomerulosclerosis resulted in distinct NSC progressions, suggesting that the NSC is suitable for investigating structure-function relations and might help unravel the unknown pathomechanisms leading to focal segmental glomerulosclerosis. The NSC is an effective algorithm for extended biophysical characterization of non-voltage-gated ion channels.

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H1Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)physiology.

Diacylglycerol-sensitive transient receptor potential (TRP) channels play crucial roles in a wide variety of biological processes and systems, but their activation mechanism is not well understood. We describe an optical toolkit by which activation and deactivation of these ion channels can be controlled with unprecedented speed and precision through light stimuli. We show that the photoswitchable diacylglycerols PhoDAG-1 and PhoDAG-3 enable rapid photoactivation of two DAG-sensitive TRP channels, Trpc2 and TRPC6, upon stimulation with UV-A light, whereas exposure to blue light terminates channel activation. PhoDAG photoconversion can be applied in heterologous expression systems, in native cells, and even in mammalian tissue slices. Combined laser scanning-controlled photoswitching and Ca2+ imaging enables both large-scale mapping of TRP channel-mediated neuronal activation and localized mapping in small cellular compartments. Light-switchable PhoDAGs provide an important advance to explore the pathophysiological relevance of DAG-sensitive TRP channels in the maintenance of body homeostasis.

Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018).

Analysis of G-protein-coupled receptor (GPCR) signaling, in particular of the second messenger cAMP that is tightly controlled by Gs- and Gi/o-proteins, is a central issue in biomedical research. The classical biochemical method to monitor increases in intracellular cAMP concentrations consists of a radioactive multicellular assay, which is well established, highly sensitive, and reproducible, but precludes continuous spatial and temporal assessment of cAMP levels in single living cells. For this purpose, Förster resonance energy transfer (FRET)-based Epac cAMP sensors are well suitable. So far, the latter sensors have been employed to monitor Gs-induced cAMP increases and it has remained elusive whether Epac sensors can reliably detect decreased intracellular cAMP levels as well. In this study, we systematically optimize experimental strategies employing FRET-based cAMP sensors to monitor Gi/o-mediated cAMP reductions. FRET experiments with adrenergic α2A or μ opioid receptors and a set of different Epac sensors allowed for time-resolved, valid, and reliable detection of cAMP level decreases upon Gi/o-coupled receptor activation in single living cells, and this effect can be reversed by selective receptor antagonists. Moreover, pre-treatment with forskolin or 3-isobutyl-1-methylxanthine (IBMX) to artificially increase basal cAMP levels was not required to monitor Gi/o-coupled receptor activation. Thus, using FRET-based cAMP sensors is of major advantage when compared to classical biochemical and multi-cellular assays.