Summary Energy production in the brain depends almost exclusively on oxidative metabolism. Neurons have small energy reserves and require a continuous supply of oxygen (O2). It is therefore not surprising that one of the hallmarks of normal brain function is the tight coupling between cerebral blood flow and neuronal activity. Since capillaries are embedded in the O2-consuming neuropil, we have here examined whether activity-dependent dips in O2 tension drive capillary hyperemia. In vivo analyses showed that transient dips in tissue O2 tension elicit capillary hyperemia. Ex vivo experiments revealed that red blood cells (RBCs) themselves act as O2 sensors that autonomously regulate their own deformability and thereby flow velocity through capillaries in response to physiological decreases in O2 tension. This observation has broad implications for understanding how local changes in blood flow are coupled to synaptic transmission.

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca 2+ ) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca 2+ influx decrease significantly. Remarkably, a critical extracellular Ca 2+ concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease. Previous studies have shown that the addition of chemicals that stiffen RBC membranes decreases the amount of ATP released (9, 16), indicating that deformation of the cell membrane is a necessary trigger. In addition, biological mediators, such as cystic fibrosis transmembrane conductance regulator (CFTR) and pannexin-1 hemichannels, are involved in the release pathways of mechanotransductive ATP release from RBCs (9,14,17,18). Inhibition of CFTR leads to an impaired ATP release from deformed RBCs (14). Recent studies, including our previous findings, suggest that interactions between membrane-associated actin and CFTR play important roles in the mechanotransductive ATP release from RBCs (9, 17). Pannexin-1, however, is a channel-forming protein and has been suggested as a mechanosensing ATP release channel (18). Under osmotic stress, for example, ATP released from RBCs was attenuated by carbenoxolone, a highly effective pannexin channel blocker, suggesting that pannexin-1 might be one of the conductance channels responsible for the mechanotransductive release of ATP (18). Although progress has been made in understanding mechanotransductive ATP release from RBCs, many questions remain about the signal transduction pathways. For example, how does mechanical force transduce signals to ATP release channels? Are there any stretch-activated ion channels on RBCs that may sense mechanical forces and activate ATP release? If so, are there any secondary messengers that could be generated by mechanical stimuli and regulate ATP release?Piezo proteins (Piezo1 and Pizeo2) are recently identified mechanically activated cation channels in mammals (19,20) and can be fully activated without involvement of additional proteins (20, 21). Piezo-induced cationic currents were first observe...

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.