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.

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.

Microshell Arrays Enhanced Sensitivity in Detection of Specific Antibody for Reduced Graphene Oxide Optical Sensor.

Protein-protein interactions play an important role in the investigation of biomolecules. In this paper, we reported on the use of a reduced graphene oxide microshell (RGOM)-based optical biosensor for the determination of goat anti-rabbit IgG. The biosensor was prepared through a self-assembly of monolayers of monodisperse polystyrene microspheres, combined with a high-temperature reduction, in order to decorate the RGOM with rabbit IgG. The periodic microshells allowed a simpler functionalization and modification of RGOM with bioreceptor units, than reduced graphene oxide (RGO). With additional antibody-antigen binding, the RGOM-based biosensor achieved better real-time and label-free detection. The RGOM-based biosensor presented a more satisfactory response to goat anti-rabbit IgG than the RGO-based biosensor. This method is promising for immobilizing biomolecules on graphene surfaces and for the fabrication of biosensors with enhanced sensitivity.

Ultrasensitive flow sensing of a single cell using graphene-based optical sensors.

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.

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells.

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca(2+)]c) and nuclear calcium ([Ca(2+)]n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca(2+)]n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca(2+)]n and [Ca(2+)]c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.

Collective Cell Radial Ordered Migration in Spatial Confinement.

Collective cells, a typical active matter system, exhibit complex coordinated behaviors fundamental for various developmental and physiological processes. The present work discovers a collective radial ordered migration behavior of NIH3T3 fibroblasts that depends on persistent top-down regulation with 2D spatial confinement. Remarkably, individual cells move in a weak-oriented, diffusive-like rather than strong-oriented ballistic manner. Despite this, the collective movement is spatiotemporal heterogeneous and radial ordering at supracellular scale, manifesting as a radial ordered wavefront originated from the boundary and propagated toward the center of pattern. Combining bottom-up cell-to-extracellular matrix (ECM) interaction strategy, numerical simulations based on a developed mechanical model well reproduce and explain above observations. The model further predicts the independence of geometric features on this ordering behavior, which is validated by experiments. These results together indicate such radial ordered collective migration is ascribed to the couple of top-down regulation with spatial restriction and bottom-up cellular endogenous nature.

Cell-cell communication induces random spikes of spontaneous calcium oscillations in multi-BV-2 microglial cells.

As the first and main form of active immune defense in the central nervous system, microglial cells usually exhibit complicated intracellular calcium (Ca²âº) activity that can regulate the downstream components of signaling cascades. In the present work, spontaneous oscillations of the cytosolic calcium concentration ([Ca²âº]c) in multi-BV-2 microglial cells were observed by video microscopy. These cells exhibited random spikes of Ca²âº oscillations. Cross-correlation analysis of the temporal dependence of the oscillations indicated the existence of cell-cell communication mediated by extracellular messengers. Numerical simulations based on a simple mathematical model suggested that these communications could induce random spikes of spontaneous Ca oscillations in the multi-cell system. Short-time imaging analysis of random spikes in different regions of a single cell showed that spontaneous Ca²âº oscillations resulted from Ca²âº wave generated by other cells as well as from calcium elevation inside the cell. Taken together, our data demonstrate that cell-cell communication existed between the BV-2 microglial cells in vitro and further resulted in the random spikes of spontaneous Ca²âº oscillations.

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.

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.

O-GlcNAcylation Regulates Centrosome Behavior and Cell Polarity to Reduce Pulmonary Fibrosis and Maintain the Epithelial Phenotype.

O-GlcNAcylation functions as a cellular nutrient and stress sensor and participates in almost all cellular processes. However, it remains unclear whether O-GlcNAcylation plays a role in the establishment and maintenance of cell polarity, because mice lacking O-GlcNAc transferase (OGT) are embryonically lethal. Here, a mild Ogt knockout mouse model is constructed and the important role of O-GlcNAcylation in establishing and maintaining cell polarity is demonstrated. Ogt knockout leads to severe pulmonary fibrosis and dramatically promotes epithelial-to-mesenchymal transition. Mechanistic studies reveal that OGT interacts with pericentriolar material 1 (PCM1) and centrosomal protein 131 (CEP131), components of centriolar satellites required for anchoring microtubules to the centrosome. These data further show that O-GlcNAcylation of PCM1 and CEP131 promotes their centrosomal localization through phase separation. Decrease in O-GlcNAcylation prevents PCM1 and CEP131 from localizing to the centrosome, instead dispersing these proteins throughout the cell and impairing the microtubule-centrosome interaction to disrupt centrosome positioning and cell polarity. These findings identify a previously unrecognized role for protein O-GlcNAcylation in establishing and maintaining cell polarity with important implications for the pathogenesis of pulmonary fibrosis.

Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation.

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) method to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution-scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Overall, we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.

Intracellular delivery of nitric oxide enhances the therapeutic efficacy of mesenchymal stem cells for myocardial infarction.

Cell therapy by autologous mesenchymal stem cells (MSCs) is a clinically acceptable strategy for treating various diseases. Unfortunately, the therapeutic efficacy is largely affected by the low quality of MSCs collected from patients. Here, we showed that the gene expression of MSCs from patients with diabetes was differentially regulated compared to that of MSCs from healthy controls. Then, MSCs were genetically engineered to catalyze an NO prodrug to release NO intracellularly. Compared to extracellular NO conversion, intracellular NO delivery effectively prolonged survival and enhanced the paracrine function of MSCs, as demonstrated by in vitro and in vivo assays. The enhanced therapeutic efficacy of engineered MSCs combined with intracellular NO delivery was further confirmed in mouse and rat models of myocardial infarction, and a clinically relevant cell administration paradigm through secondary thoracotomy has been attempted.

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.

Nondegradable ubiquitinated ATG9A organizes Golgi integrity and dynamics upon stresses.

ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.

A multishear microfluidic device for quantitative analysis of calcium dynamics in osteoblasts.

Microfluidics is a convenient platform to study the influences of fluid shear stress on calcium dynamics. Fluidic shear stress has been proven to affect bone cell functions and remodelling. We have developed a microfluidic system which can generate four shear flows in one device as a means to study cytosolic calcium concentration ([Ca(2+)](c)) dynamics of osteoblasts. Four shear forces were achieved by having four cell culture chambers with different widths while resistance correction channels compensated for the overall resistance to allow equal flow distribution towards the chambers. Computational simulation of the local shear stress distribution highlighted the preferred section in the cell chamber to measure the calcium dynamics. Osteoblasts showed an [Ca(2+)](c) increment proportional to the intensity of the shear stress from 0.03 to 0.30 Pa. A delay in response was observed with an activation threshold between 0.03 and 0.06 Pa. With computational modelling, our microfluidic device can offer controllable multishear stresses and perform quantitative comparisons of shear stress-induced intensity change of calcium in osteoblasts.

Magnetic fields at extremely low-frequency (50 Hz, 0.8 mT) can induce the uptake of intracellular calcium levels in osteoblasts.

Several studies have been undertaken to elucidate the effects of electromagnetic field (EMF) on intracellular calcium ([Ca(2+)](i)) in the past 20 years. However, still there were controversies of electromagnetic pollution within the scientific community. In this work, we studied the effects of alternative magnetic fields on intracellular calcium. Osteoblastic cells were used as a model both to test the hypothesis that extremely low-frequency (ELF) magnetic fields can alter the concentrations of the intracellular calcium, and to examine the 'window' effect predicted by our previous theoretical work. The outcome of this experiment demonstrated that 50 Hz, 0.8 mT magnetic field can induce the uptake of [Ca(2+)](i) in osteoblasts. The empirical evidences of the specified window effects of [Ca(2+)](i) in osteoblastic cells were reported for the first time in this work.

Ultraviolet irradiation induces autofluorescence enhancement via production of reactive oxygen species and photodecomposition in erythrocytes.

Ultraviolet (UV) light has a significant influence on human health. In this study, human erythrocytes were exposed to UV light to investigate the effects of UV irradiation (UVI) on autofluorescence. Our results showed that high-dose continuous UVI enhanced erythrocyte autofluorescence, whereas low-dose pulsed UVI alone did not have this effect. Further, we found that H(2)O(2), one type of reactive oxygen species (ROS), accelerated autofluorescence enhancement under both continuous and pulsed UVI. In contrast, continuous and pulsed visible light did not result in erythrocyte autofluorescence enhancement in the presence or absence of H(2)O(2). Moreover, NAD(P)H had little effect on UVI-induced autofluorescence enhancement. From these studies, we conclude that UVI-induced erythrocyte autofluorescence enhancement via both UVI-dependent ROS production and photodecomposition. Finally, we present a theoretical study of this autofluorescence enhancement using a rate equation model. Notably, the results of this theoretical simulation agree well with the experimental data further supporting our conclusion that UVI plays two roles in the autofluorescence enhancement process.

FZR1 as a novel biomarker for breast cancer neoadjuvant chemotherapy prediction.

The concept of breast-conserving surgery is a remarkable achievement of breast cancer therapy. Neoadjuvant chemotherapy is being used increasingly to shrink the tumor prior to surgery. Neoadjuvant chemotherapy is reducing the tumor size to make the surgery with less damaging to surrounding tissue and downstage locally inoperable disease to operable. However, non-effective neoadjuvant chemotherapy could increase the risks of delaying surgery, develop unresectable disease and metastatic tumor spread. The biomarkers for predicting the neoadjuvant chemotherapy effect are scarce in breast cancer treatment. In this study, we identified that FZR1 can be a novel biomarker for breast cancer neoadjuvant chemotherapy according to clinical patient cohort evaluation and molecular mechanism investigation. Transcriptomic data analysis indicated that the expression of FZR1 is correlated with the effect of neoadjuvant chemotherapy. Mechanistically, we demonstrate that FZR1 is pivotal to the chemotherapy drugs induced apoptosis and cell cycle arrest. FZR1 is involved in the stability of p53 by impairing the phosphorylation at ser15 site. We demonstrate that the expression of FZR1 detected by quantification of IHC can be an effective predictor of neoadjuvant chemotherapy in animal experiment and clinical patient cohort. To obtain more benefit for breast cancer patient, we propose that the FZR1 IHC score using at the clinical to predict the effect of neoadjuvant chemotherapy.

Optical trapping and manipulation of metallic micro/nanoparticles via photorefractive crystals.

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.