DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden.

The P2X7 receptor (P2X7R) stands out within the purinergic family as it has exclusive pharmacological and regulatory features, and it fulfills distinct roles depending on the type of stimulation and cellular environment. Tonic activation of P2X7R promotes cell proliferation, whereas sustained activation is associated with cell death. Yet strikingly, prolonged P2X7R activation in rat cerebellar granule neurons and astrocytes does not affect cell survival. The intracellular pathways activated by P2X7Rs involve proteins like MAPKs, ERK1/2 and p38, and interactions with growth factor receptors could explain their behavior in populations of rat cerebellar cells. In this study, we set out to characterize the intracellular mechanisms through which P2X7Rs and Trk receptors, EGFR (epidermal growth factor receptor) and BDNFR (brain-derived neurotrophic factor receptor), regulate the dual-specificity phosphatase DUSP1. In cerebellar astrocytes, the regulation of DUSP1 expression by P2X7R depends on ERK and p38 activation. EGFR stimulation can also induce DUSP1 expression, albeit less strongly than P2X7R. Conversely, EGF was virtually ineffective in regulating DUSP1 in granule neurons, a cell type in which BDNF is the main regulator of DUSP1 expression and P2X7R only induces a mild response. Indeed, the regulation of DUSP1 elicited by BDNF reflects the balance between both transcriptional and post-transcriptional mechanisms. Importantly, when the regulation of DUSP1 expression is compromised, the viability of both astrocytes and neurons is impaired, suggesting this phosphatase is essential to maintain proper cell cytoarchitecture and functioning.

Single-Molecule Analysis of Genome Uncoating from Individual Human Rhinovirus Particles, and Modulation by Antiviral Drugs.

Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single-molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force-induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA-free capsids, led to a lower force-induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high-force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol-1 ), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.

Measuring (biological) materials mechanics with atomic force microscopy. 5. Traction force microscopy (cell traction forces).

Cells generate traction forces to probe the mechanical properties of the surroundings and maintain a basal equilibrium state of stress. Traction forces are also implicated in cell migration, adhesion and ECM remodeling, and alteration of these forces is often observed in pathologies such as cancer. Thus, analyzing the traction forces is important for studies of cell mechanics in cancer and metastasis. In this primer, the methodology for conducting two-dimensional traction force microscopy (2D-TFM) experiments is reported. As a practical example, we analyzed the traction forces generated by three human breast cancer cell lines of different metastatic potential: MCF10-A, MCF-7 and MDA-MB-231 cells, and studied the effects of actin cytoskeleton disruption on those traction forces. Contrary to what is often reported in literature, lower traction forces were observed in cells with higher metastatic potential (MDA-MB-231). Implications of substrate stiffness and concentration of extracellular matrix proteins in such findings are discussed in the text. RESEARCH HIGHLIGHTS: Traction force microscopy (TFM) is suitable for studying and quantifying cell-substrate and cell-cell forces. TFM is suitable for investigating the relationship between chemical to mechanical signal transduction and vice versa. TFM can be combined with classical indentation studies providing a compact picture of cell mechanics. TFM still needs new physico-chemical (sample preparation) and computational approaches for more accurate data evaluation.

Nucleotide-Induced Nanoscale Changes in the Mechanical Properties of Rat Cerebellar Astrocytes: Selective Stimulation and Blocking of the Purinergic Receptor P2X7.

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.

Measuring biological materials mechanics with atomic force microscopy - Mechanical unfolding of biopolymers.

Biopolymers, such as polynucleotides, polypeptides and polysaccharides, are macromolecules that direct most of the functions in living beings. Studying the mechanical unfolding of biopolymers provides important information about their molecular elasticity and mechanical stability, as well as their energy landscape, which is especially important in proteins, since their three-dimensional structure is essential for their correct activity. In this primer, we present how to study the mechanical properties of proteins with atomic force microscopy and how to obtain information about their stability and energetic landscape. In particular, we discuss the preparation of polyprotein constructs suitable for AFM single molecule force spectroscopy (SMFS), describe the parameters used in our force-extension SMFS experiments and the models and equations employed in the analysis of the data. As a practical example, we show the effect of the temperature on the unfolding force, the distance to the transition state, the unfolding rate at zero force, the height of the transition state barrier, and the spring constant of the protein for a construct containing nine repeats of the I27 domain from the muscle protein titin. HIGHLIGHTS: 1. Atomic force microscopy (AFM) can be used to study the mechanical unfolding of polymers. 2. AFM provides a direct measurement of unfolding (unbinding) forces. 3. Force measurements for different rates provide information about the distance to the transition state and the unfolding rate at zero force.

Substrate stiffness modulates the viscoelastic properties of MCF-7 cells.

Cells sense stiffness of surrounding tissues and adapt their activity, proliferation, motility and mechanical properties based on such interactions. Cells probe the stiffness of the substrate by anchoring and pulling to their surroundings, transmitting force to the extracellular matrix and other cells, and respond to the resistance they sense, mainly through changes in their cytoskeleton. Cancer and other diseases alter stiffness of tissues, and the response of cancer cells to this stiffness can also be affected. In the present study we show that MCF-7 breast cancer cells seeded on polyacrylamide gels have the ability to detect the stiffness of the substrate and alter their mechanical properties in response. MCF-7 cells plated on soft substrates display lower stiffness and viscosity when compared to those seeded on stiffer gels or glass. These differences can be associated with differences in the morphology and cytoskeleton organisation, since cells seeded on soft substrates have a round morphology, while cells seeded on stiffer substrates acquire a flat and spread morphology with formation of actin filaments, similar to that observed when seeded on glass. These findings show that MCF-7 cells can detect the stiffness of the surrounding microenvironment and thus, modify their mechanical properties.

BCI, an inhibitor of the DUSP1 and DUSP6 dual specificity phosphatases, enhances P2X7 receptor expression in neuroblastoma cells.

P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expression, whereas it induced a decrease in the dual specificity phosphatase 6 transcripts, suggesting that BCI-dependent inhibition of dual specificity phosphatase 1 may be responsible for the increase in p38 and JNK phosphorylation. The weaker ERK phosphorylation induced by BCI was reversed by p38 inhibition, indicating that this MAPK is involved in the regulatory loop that dampens ERK activity. The PP2A phosphatase appears to be implicated in the p38-dependent dephosphorylation of ERK1/2. In addition, the PTEN phosphatase inhibition also prevented ERK1/2 dephosphorylation, probably through p38 downregulation. By contrast, inhibition of the p53 nuclear factor decreased ERK phosphorylation, probably enhancing the activity of p38. Finally, the inhibition of either p38 or Sp1-dependent transcription halved the increase in P2X7 receptor expression induced by BCI. Moreover, the combined inhibition of both p38 and Sp1 completely prevented the effect exerted by BCI. Together, our results indicate that dual specificity phosphatase 1 acts as a novel negative regulator of P2X7 receptor expression in neuroblastoma cells due to the downregulation of the p38 pathway.

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy.

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.

Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells.

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to DUSP transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation. Potent DUSP gene inducers also include cannabinoids, which preserve DUSP activity in inflammatory conditions. Additionally, nucleotides activating P2X7 and P2Y13 nucleotide receptors behave as novel players in the regulation of DUSP function. They increase cell survival in stressful conditions, regulating DUSP protein turnover and inducing DUSP gene expression. In general terms, in the context of neural cells exposed to damaging conditions, the recovery of DUSP activity is neuroprotective and counteracts pro-apoptotic over-activation of p38 and JNK. In addition, remarkable changes in DUSP function take place during the onset of neuropathologies. The restoration of proper DUSP levels and recovery of MAPK homeostasis underlie the therapeutic effect, indicating that DUSPs can be relevant targets for brain diseases.

P2 receptor interaction and signalling cascades in neuroprotection.

Nucleotides can contribute to the survival of different glial and neuronal models at the nervous system via activation of purinergic P2X and P2Y receptors. Their activation counteracts different proapoptotic events, such as excitotoxicity, mitochondrial impairment, oxidative stress and DNA damage, which concur to elicit cell loss in different processes of neurodegeneration and brain injury. Thus, it is frequent to find that different neuroprotective mediators converge in the activation of the same intracellular survival pathways to protect cells from death. The present review focuses on the role of P2Y1 and P2Y13 metabotropic receptors, and P2X7 ionotropic receptors to regulate the balance between survival and apoptosis. In particular, we analyze the intracellular pathways involved in the signaling of these nucleotide receptors to elicit survival, including calcium/PLC, PI3K/Akt/GSK3, MAPK cascades, and the expression of antioxidant and antiapoptotic genes. This review emphasizes the novel contribution of nucleotide receptors to maintain cell homeostasis through the regulation of MAP kinases and phosphatases. Unraveling the different roles found for nucleotide receptors in different models and cellular contexts may be crucial to delineate future therapeutic applications based on targeting nucleotide receptors for neuroprotection.

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.