Jiandi Wan

Piezo1 regulates mechanotransductive release of ATP from human RBCs

Significance Mechanotransductive release of ATP from RBCs participates in the regulation of microvascular tone and plays essential roles in vascular physiopathology. The mechanism responsible for the release of ATP, however, is poorly understood. We show for the first time, to our knowledge, that Piezo1, the recently identified mechanically activated cation channel, regulates the mechanotransductive release of ATP from RBCs. In particular, we uncover the link between Piezo1, calcium influx, and ATP release and propose a previously unidentified mechanotransductive pathway that modulates the release of ATP from RBCs. The outcome of this study will allow the development of much needed approaches to regulate the release of ATP from RBCs and thus, impact significantly the fields of red cell research, cellular mechanosensing, and Piezo1 channels. 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 (Ca2+) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca2+ influx decrease significantly. Remarkably, a critical extracellular Ca2+ 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.

Bio-printing cell-laden Matrigel–agarose constructs

3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel–agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel–agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel–agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering.

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells.

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

Significance The biconcave disk shape and deformability of the mammalian RBC are vital to its circulatory function and rely upon a 2D viscoelastic spectrin–F-actin network attached to the membrane. A role for nonmuscle myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has not been tested. We show that NMIIA forms bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity regulates interactions with the spectrin–F-actin network to control RBC biconcave shape and deformability. These results provide a previously undescribed mechanism for actomyosin force generation at the plasma membrane, and may apply to spectrin–F-actin–based membrane skeleton networks in other cell types, such as neurons and polarized epithelial cells. 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.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Evaporation-based microfluidic production of oil-free cell-containing hydrogel particles

We demonstrate an evaporation-based microfluidic strategy to produce oil-free cell containing hydrogel particles. Perfluoro-n-pentane, which is used as the continuous oil phase to generate cell-containing hydrogel (Extracel) particles, is removed at an elevated temperature. Human colon cancer cells (HCT116) encapsulated in the hydrogel particles show higher viability than cells encapsulated in particles that are produced via a non-evaporative oil phase. In addition, single HCT116 cells can be cultured for a week in such particles and respond to inflammatory stimuli, highlighting the potential applications of the developed strategy for 3D cell culture, drug testing, and cell-based drug delivery.

Enhanced separation of aged RBCs by designing channel cross section

Prolonged storage will alter the biophysical properties of red blood cells (RBCs), and it decreases the quality of stored blood for blood transfusion. It has been known that less deformable aged RBCs can be separated by margination, but the recognition of the storage time from the separation efficiency of the stiff RBCs is still a challenge. In this study, we realized enhanced separation of aged RBCs from normal RBCs by controlling the channel cross section and demonstrated that the storage time can be deduced from the percentage of the separated RBCs in the stored RBCs. This separation technology helps to reveal the regulation of time on the RBC aging mechanism and offer a new method to separate stiffened cells with high efficiency.

Microfluidic assay of the deformability of primitive erythroblasts

Primitive erythroblasts (precursors of red blood cells) enter vascular circulation during the embryonic period and mature while circulating. As a result, primitive erythroblasts constantly experience significant hemodynamic shear stress. Shear-induced deformation of primitive erythroblasts however, is poorly studied. In this work, we examined the deformability of primitive erythroblasts at physiologically relevant flow conditions in microfluidic channels and identified the regulatory roles of the maturation stage of primitive erythroblasts and cytoskeletal protein 4.1 R in shear-induced cell deformation. The results showed that the maturation stage affected the deformability of primitive erythroblasts significantly and that primitive erythroblasts at later maturational stages exhibited a better deformability due to a matured cytoskeletal structure in the cell membrane.

In vitro microfluidic circulatory system for circulating cancer cells

Circulating tumor cells (CTCs) experience hemodynamic shear stress in circulation and play critical roles in cancer metastasis. The effect of shear on CTCs, however, remains less studied. Here, we described a protocol to circulate HCT116 human colon cancer cells in a microfluidic circulatory system mimicking physiologically relevant circulating conditions. This protocol represents a useful scaffold to mimic the transportation of CTCs in circulation and thus provides an effective means to study the effect of shear on CTCs. We anticipate that future studies using the developed system will help us to further investigate the regulatory roles of shear in molecular responses of CTCs.