The erythrocyte cytoskeleton is a textbook prototype for the submembrane cytoskeleton of metazoan cells. While early experiments suggest a triangular network of actin-based junctional complexes connected by ∼200-nm-long spectrin tetramers, later studies indicate much smaller junction-to-junction distances in the range of 25-60 nm. Through super-resolution microscopy, we resolve the native ultrastructure of the cytoskeleton of membrane-preserved erythrocytes for the N and C termini of β-spectrin, F-actin, protein 4.1, tropomodulin, and adducin. This allows us to determine an ∼80-nm junction-to-junction distance, a length consistent with relaxed spectrin tetramers and theories based on spectrin abundance. Through two-color data, we further show that the cytoskeleton meshwork often contains nanoscale voids where the cell membrane remains intact and that actin filaments and capping proteins localize to a subset of, but not all, junctional complexes. Together, our results call for a reassessment of the structure and function of the submembrane cytoskeleton.

On the basis of the polarization-dependent absorption of graphene under total internal reflection, we designed a graphene-based optical refractive index sensor with high resolution of 1.7 × 10(-8) and sensitivity of 4.3 × 10(7) mV/RIU, as well as an extensive dynamic range. This highly sensitive graphene optical sensor enables label-free, live-cell, and highly accurate detection of a small quantity of cancer cells among normal cells at the single-cell level and the simultaneous detection and distinction of two cell lines without separation. It provides an accurate statistical distribution of normal and cancer cells with fewer cells. This facile and highly sensitive sensing refractive index may expand the practical applications of the biosensor.

A simple method to trap and manipulate metallic micro/nano-particles on the surface of photorefractive crystals is proposed. After inducing inhomogeneous charge density and space-charge fields in photorefractive crystals by non-uniform illumination, both uncharged and charged metallic particles can be trapped on the illuminated surface due to dielectrophoretic force and electrophoretic force, respectively. A transition from dielectrophoresis to electrophoresis is observed when manipulating nano-silver particles with high surface space-charge field. Our results show that this method is simple and effective to form surface microstructures of metallic particles.

AimsThe local concentration of extracellular Ca2+ ([Ca2+]o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca2+]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca2+]o to osteoblastic proliferation.MethodsCytosolic Ca2+ concentration ([Ca2+]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.ResultsOur data showed that elevating [Ca2+]o evoked a sustained increase of [Ca2+]c in a dose-dependent manner. This [Ca2+]c increase was blocked by TMB-8 (Ca2+ 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 [Ca2+]o-induced [Ca2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca2+]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.ConclusionsElevating [Ca2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca2+ concentration.

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Rebuilding injured tissue for regenerative medicine requires technologies to reproduce tissue/biomaterials mimicking the natural morphology. To reconstitute the tissue pattern, current approaches include using scaffold with specific structure to plate cells, guiding cell spreading, or directly moving cells to desired locations. However, the structural complexity is limited. Also, the artificially-defined patterns are usually disorganized by cellular self-organization in the subsequent tissue development, such as cell migration and cell-cell communication. Here, by working in concert with cellular self-organization rather than against it, we experimentally and mathematically demonstrate a method which directs self-organizing vascular mesenchymal cells (VMCs) to assemble into desired multicellular patterns. Incorporating the inherent chirality of VMCs revealed by interfacing with micro-engineered substrates and VMCs’ spontaneous aggregation, difference in distribution of initial cell plating can be amplified into the formation of exquisite radial structures or concentric rings mimicking the cross-sectional structure of liver lobules or osteons, respectively. Furthermore, when co-cultured with VMCs, non-pattern-forming endothelial cells tracked along the VMCs and formed a coherent radial or ring pattern in a coordinated manner, indicating the applicability to heterotypical cell organization.

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.