Super-resolution microscopy unveils the nanoscale organization and self-limiting clustering of CD47 in human erythrocytes.

The transmembrane protein CD47, an innate immune checkpoint protein, plays a pivotal role in preventing healthy erythrocytes from immune clearance. Our study utilized stochastic optical-reconstruction microscopy (STORM) and single-molecule analysis to investigate the distribution of CD47 on the human erythrocyte membrane. Contrary to previous findings in mouse erythrocytes, we discovered that CD47 exists in randomly distributed monomers rather than in clusters across the human erythrocyte membrane. Using 2nd antibody-induced crosslinking, we found that CD47 aggregates into stable clusters within minutes. By comparing these STORM results with those of the fully mobile protein CD59 and the cytoskeleton-bound membrane protein glycophorin C under similar conditions, as well as devising two-color STORM co-labeling and co-clustering experiments, we further quantitatively revealed an intermediate, self-limiting clustering behavior of CD47, elucidating its fractional (∼14%) attachment to the cytoskeleton. Moreover, we report reductions in both the amount of CD47 and its clustering capability in aged erythrocytes, providing new insight into erythrocyte senescence. Together, the combination of STORM and 2nd antibody-based crosslinking unveils the unique self-limiting clustering behavior of CD47 due to its fractional cytoskeleton attachment.

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.

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.

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.

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.

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.

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.

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.

Hypotonic Stress Induces Fast, Reversible Degradation of the Vimentin Cytoskeleton via Intracellular Calcium Release.

The dynamic response of the cell to osmotic changes is critical to its physiology and is widely exploited for cell manipulation. Here, using three-dimensional stochastic optical reconstruction microscopy (3D-STORM), a super-resolution technique, the hypotonic stress-induced ultrastructural changes of the cytoskeleton of a common fibroblast cell type are examined. Unexpectedly, these efforts lead to the discovery of a fast, yet reversible dissolution of the vimentin intermediate filament system that precedes ultrastructural changes of the supposedly more dynamic actin and tubulin cytoskeletal systems as well as changes in cell morphology. In combination with calcium imaging and biochemical analysis, it is shown that the vimentin-specific fast cytoskeletal degradation under hypotonic stress is due to proteolysis by the calcium-dependent protease calpain. The process is found to be activated by the hypotonic stress-induced calcium release from intracellular stores, and is therefore efficiently suppressed by inhibiting any part of the IP3-Ca2+-calpain pathway established in this study. Together, these findings highlight an unexpected, fast degradation mechanism for the vimentin cytoskeleton in response to external stimuli, and point to the significant, yet previously overlooked physiological impacts of hypotonic stress-induced intracellular calcium release on cell ultrastructure and function.

Rhein inhibits ATP-triggered inflammatory responses in rheumatoid rat fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disorder, which may lead to joint disabilities. So far the pathogenesis of RA remains largely undetermined, and there are still no potent drugs for clinical treatment. Rhein, a natural bioactive anthraquinone derivative, exhibited significant anti-inflammatory activities demonstrated by previous studies. Here we aimed to investigate the effects of rhein on ATP-induced inflammation responses in fibroblast-like synoviocytes isolated from a rat model of collagen induced arthritis (CIA). Our results showed that ATP triggered rapid cytosolic calcium concentration ([Ca2+]c) increase depending on extracellular Ca2+ entry. Given the major P2 subtypes expressed in rat synoviocytes were P2X4 and P2Y2 receptors, ATP-elicited calcium entry should be mainly resulted from activating P2X4. Interestingly, rhein could effectively block the ATP-induced [Ca2+]c increases in a dose-dependent manner. Besides, rhein also suppressed the production of intracellular reactive oxygen species (ROS) induced by ATP in synoviocytes that was resulted from P2X4-mediated Ca2+ entry. Brilliant blue G (BBG), which can block P2X4 receptor at high concentration, showed similar suppressive effects on above responses. Furthermore, in lipopolysaccharide-primed cells, application of ATP synergistically promoted the gene expression of cyclooxygenase-2, interleukin-6 and matrix metalloproteinase-9. Both rhein and BBG attenuated these inflammatory gene expressions enhanced by ATP. Above data together suggested a potential anti-arthritic role of rhein by inhibiting ATP-induced [Ca2+]c increase, ROS production and inflammatory gene expression targeting P2X4 in CIA rat synoviocytes, which will provide a novel insight in the therapy of RA.

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.

Super-Resolution Microscopy Reveals the Native Ultrastructure of the Erythrocyte Cytoskeleton.

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 ß-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.

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.

Protection of the biconcave profile of human erythrocytes against osmotic damage by ultraviolet-A irradiation through membrane-cytoskeleton enhancement.

To perform various physiological functions, erythrocytes possess a unique biconcave shape provided by a special architecture of the membrane-skeleton system. In the present work, we use a simple irradiation method to treat human erythrocytes with 365 nm ultraviolet-A (UVA) light at the single-cell level in vitro. Depending on the irradiation dose, UVA show protection of the biconcave profile against the detrimental action of distilled water. This protective effect can also be confirmed for saponin that damages the membrane-skeleton by vesiculation and pore formation. Interestingly, at two irradiation doses of UVA pretreatment, erythrocytes still seem to exhibit cell viability as tested by trypan blue assay even if distilled water or saponin is added. The oxidants hydrogen peroxide and cumene hydroperoxide partly simulate the protective effects. Taken together, these results demonstrate that 365 nm UVA irradiation can protect the biconcave profile of human erythrocytes through membrane-skeleton enhancement associated with a production of oxidants.

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.