Mesenchymal Migration on Adhesive-Nonadhesive Alternate Surfaces in Macrophages.

Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein-repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive-nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive-nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive-nonadhesive alternate substrates in macrophages.

Molecular Resolution Mapping of Erythrocyte Cytoskeleton by Ultrastructure Expansion Single-Molecule Localization Microscopy.

The combination of expansion microscopy and single-molecule localization microscopy has the potential to approach the molecular resolution. However, this combination meets challenges due to the hydrogel shrinkage in the presence of imaging buffer. Here, a method of ultrastructure expansion single-molecule localization microscopy (U-ExSMLM) based on skillfully adhering the gel onto poly-l-lysine (pLL)-coated coverslip is developed to prevent lateral shrinkage of the hydrogel. U-ExSMLM is then applied to dissect the membrane cytoskeleton organization of human erythrocytes at molecular resolution. The resolved nanoscale spatial distributions of cytoskeleton proteins, including the N/C-termini of ß-spectrin, protein 4.1, and tropomodulin, show good agreement with the acknowledged model of erythrocyte cytoskeleton structure, demonstrating the reliability of U-ExSMLM. Furthermore, the concentration of pLL is adjusted to preserve the physiological biconcave morphology of erythrocytes, and it is found that the spectrin cytoskeleton in the dimple regions has lower density and larger length than that in the rim regions, which provides the direct evidence for cytoskeleton asymmetry in human erythrocytes. Therefore, the integrated method offers future opportunities to study the ultrastructure of membrane cytoskeleton at molecular resolution.

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages.

Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50 nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes at the layer of myosin IIA. The microtubule disruption-caused podosome dissolution is previously ascribed to Rho/ROCK-myosin signaling, yet inhibiting this pathway with Y27632 or blebbistatin only partially recovers podosome assembly, thus suggesting the contribution of the physical supporting of microtubules in stabilizing podosome structures. Through improved substrate-coating technique, we further corroborate that the matrix-degrading capability of macrophages depends on the formation of podosome clusters. Together, 3D-STORM super-resolution microscopy reveals the nanoscale spatial arrangement and the microtubule-dependent regulation of the matrix-degrading podosome clusters in macrophages.

Intercellular Bridge Mediates Ca2+ Signals between Micropatterned Cells via IP3 and Ca2+ Diffusion.

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca2+ communication between postmitotic cells. In this work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca2+ signal transfer over the intercellular bridges. With the use of such micropatterns, considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular-bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response, demonstrated by application of Ca2+-free medium, exogenous ATP, and P2Y13 receptor antagonist. In contrast, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-aminoethyl diphenylborate. Those observations prove that both passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular-bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.

Microfluidic assemblies designed for assessment of drug effects on deformability of human erythrocytes.

Extreme deformability of human erythrocytes is a prerequisite for their ability to squeeze through narrow capillaries of the blood microcirculation system. Various drugs can modify this deformability and consequently provoke circulation problems. We demonstrate that microfluidic assemblies are very convenient platforms for in vitro study of the associated processes. Two types of microfluidic channels were designed to quantitatively investigate modifications of erythrocyte deformability induced by hydrogen peroxide, ethanol and pentoxifylline based on transit velocity measurements. With a high sensitivity our microfluidic assemblies show that hydrogen peroxide decreases erythrocyte deformability in a dose-dependent manner. Then, results on ethanol resolve a biphasic nature of this reactant on the deformability of single erythrocyte cells. Results on pentoxifylline provide evidence that, similar to ethanol, also this medical drug has a double-sided effect on the erythrocyte deformability, i.e. increasing the deformability at low concentrations, while decreasing it at higher ones. Taken together, our microfluidic designs propose a potent measurement method for the erythrocyte deformability, as well as providing a perspective to evaluate effects of drugs on it.

Spatiotemporal Characteristics of Intercellular Calcium Wave Communication in Micropatterned Assemblies of Single Cells.

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 µm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity.

Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.

Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 µM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts.

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca2+]c) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca2+ entry through activation of TRPV4 channel in synoviocytes.

Rhein antagonizes P2X7 receptor in rat peritoneal macrophages.

P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca(2+)]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1ß release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca(2+)]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1ß release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.

Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.

AIMS: The local concentration of extracellular Ca(2+) ([Ca(2+)]o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca(2+)]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca(2+)]o to osteoblastic proliferation. METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)]c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail. RESULTS: Our data showed that elevating [Ca(2+)]o evoked a sustained increase of [Ca(2+)]c in a dose-dependent manner. This [Ca(2+)]c increase was blocked by TMB-8 (Ca(2+) release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca(2+)]o-induced [Ca(2+)]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca(2+)]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca(2+)]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. CONCLUSIONS: Elevating [Ca(2+)]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca(2+) concentration.