Function of membrane domains in rho-of-plant signaling

In a crowded environment, establishing interactions between different molecular partners can take a long time. Biological membranes have solved this issue, as they simultaneously are fluid and possess compartmentalized domains. This nanoscale organization of the membrane is often based on weak, local, and multivalent interactions between lipids and proteins. However, from local interactions at the nanoscale, different functional properties emerge at the higher scale, and these are critical to regulate and integrate cellular signaling. Rho of Plant (ROP) proteins are small guanosine triphosphate hydrolase enzymes (GTPases) involved in hormonal, biotic, and abiotic signaling, as well as fundamental cell biological properties such as polarity, vesicular trafficking, and cytoskeleton dynamics. Association with the membrane is essential for ROP function, as well as their precise targeting within micrometer-sized polar domains (i.e. microdomains) and nanometer-sized clusters (i.e. nanodomains). Here, we review our current knowledge about the formation and the maintenance of the ROP domains in membranes. Furthermore, we propose a model for ROP membrane targeting and discuss how the nanoscale organization of ROPs in membranes could determine signaling parameters like signal specificity, amplification, and integration.

The nanoscale organization of Rho of Plant proteins creates emergent properties that determine cellular signaling.     

1950 MHz射频电磁场对SRA01/04细胞周期和凋亡的影响

目的：研究显示射频电磁场与白内障的发生关系密切,为了评价晶状体上皮细胞在射频电磁场诱导的白内障发生中的作 用,本实验探讨了1950 MHz射频电磁场暴露对人眼晶状体上皮细胞株（SRA01/04）细胞周期与凋亡的影响。方法：将处于对数生 长期的SRA01/04 细胞暴露或假暴露于频率为1950 MHz,比吸收率（SAR）为2.79 W/kg 的射频电磁场中,每天暴露1 h,每周暴露 5 天,连续暴露4 周。暴露结束后立即收集细胞,显微镜下观察细胞形态变化,噻唑蓝（MTT）法检测细胞存活力,流式细胞仪 （FCM）检测细胞周期与凋亡。结果：与假辐照组相比,暴露组细胞形态未见明显变化;细胞存活力、细胞周期分布及细胞凋亡率亦 无显著改变（P>0.05）。结论：1950 MHz射频电磁场暴露4 周对SRA01/04 细胞的形态、活力、周期以及凋亡均无明显影响,提示在 本实验条件下1950 MHz 射频电磁场不会诱发白内障的发生。     

Biophysical limits on athermal effects of RF and microwave radiation

Using biophysical criteria, I show that continuous radiofrequency (RF) and microwave radiation with intensity less than 10 mW/cm(2) are unlikely to affect physiology significantly through athermal mechanisms. Biological systems are fundamentally noisy on the molecular scale as a consequence of thermal agitation and are noisy macroscopically as a consequence of physiological functions and animal behavior. If electromagnetic fields are to significantly affect physiology, their direct physical effect must be greater than that from the ubiquitous endogenous noise. Using that criterion, I show that none of a set of interactions of weak fields, which I argue is nearly complete on dimensional grounds, can affect biology on the molecular scale. Moreover, I conclude that such weak fields are quite unlikely to generate significant effects in their interactions with larger biological elements such as cells. In the course of that analysis, I examine important special examples of electromagnetic interactions: "direct" interactions where biology is modified simply by the motion of charged elements generated by the electric field; resonance interactions; the effects of electrostrictive forces and induced dipole moments; and modifications of radical pair recombination probabilities. In each case, I show that it is unlikely that low intensity fields can generate significant physiological consequences.     

Exposure to 900 MHz electromagnetic field induces an unbalance between pro-apoptotic and pro-survival signals in T-lymphoblastoid leukemia CCRF-CEM cells

It has been recently established that low-frequency electromagnetic field (EMFs) exposure induces biological changes and could be associated with increased incidence of cancer, while the issue remains unresolved as to whether high-frequency EMFs can have hazardous effect on health. Epidemiological studies on association between childhood cancers, particularly leukemia and brain cancer, and exposure to low- and high-frequency EMF suggested an etiological role of EMFs in inducing adverse health effects. To investigate whether exposure to high-frequency EMFs could affect in vitro cell survival, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of unmodulated 900 MHz EMF, generated by a transverse electromagnetic (TEM) cell, at various exposure times. We evaluated the effects of high-frequency EMF on cell growth rate and apoptosis induction, by cell viability (MTT) test, FACS analysis and DNA ladder, and we investigated pro-apoptotic and pro-survival signaling pathways possibly involved as a function of exposure time by Western blot analysis. At short exposure times (2-12 h), unmodulated 900 MHz EMF induced DNA breaks and early activation of both p53-dependent and -independent apoptotic pathways while longer continuous exposure (24-48 h) determined silencing of pro-apoptotic signals and activation of genes involved in both intracellular (Bcl-2) and extracellular (Ras and Akt1) pro-survival signaling. Overall our results indicate that exposure to 900 MHz continuous wave, after inducing an early self-defense response triggered by DNA damage, could confer to the survivor CCRF-CEM cells a further advantage to survive and proliferate.     

Alteration of tight and adherens junctions on 50-Hz magnetic field exposure in Madin Darby canine kidney (MDCK) cells

Adherens (AJ) and tight junctions (TJ), as integrated parts of the junctional complex, are multifunctional specialized regions of the cell membrane in epithelial cells. They are responsible for cell-to-cell interactions and also have great importance in cellular signaling processes including Wnt protein-mediated signals. As electromagnetic field (EMF) exposure is known to cause alterations in the function as well as supramolecular organization of different cell contacts, our goal was to investigate the effect of 50-Hz magnetic field (MF) exposures on the subcellular distribution of some representative structural proteins (occludin, beta-catenin, and cadherin) found in AJ and TJ. Additionally, cellular beta-catenin content was also quantified by Western blot analysis. 50-Hz MF exposures seemed to increase the staining intensity (amount) of occludin, cadherins, and beta-catenin in the junctional area of MDCK cells, while Western blot data indicated the quantity of beta-catenin was found significantly decreased at both time points after EM exposures. Our results demonstrate that MF are able to modify the distribution of TJ and AJ structural proteins, tending to stabilize these cell contacts. The quantitative changes of beta-catenin suggest a causative relationship between MF effects on the cell junctional complex and the Wnt signaling pathway.     

1950MHz射频电磁场对SRA01／04细胞周期和凋亡的影响

目的：研究显示射频电磁场与白内障的发生关系密切,为了评价晶状体上皮细胞在射频电磁场诱导的白内障发生中的作用,本实验探讨了1950MHz射频电磁场暴露对人眼晶状体上皮细胞株（SRA01／04）细胞周期与凋亡的影响。方法：将处于对数生长期的SRA01／04细胞暴露或假暴露于频率为1950MHz,比吸收率（SAR）为2．79W／kg的射频电磁场中,每天暴露1h,每周暴露5天,连续暴露4周。暴露结束后立即收集细胞,显微镜下观察细胞形态变化,噻唑蓝（MTT）法检测细胞存活力,流式细胞仪（FCM）检测细胞周期与凋亡。结果：与假辐照组相比,暴露组细胞形态未见明显变化;细胞存活力、细胞周期分布及细胞凋亡率亦无显著改变（P〉0．05）。结论：1950MHz射频电磁场暴露4周对SRA01／04细胞的形态、活力、周期以及凋亡均无明显影响,提示在本实验条件下1950MHz射频电磁场不会诱发白内障的发生。     

Theoretical Simulation and Focused Ion Beam Fabrication of Gold Nanostructures for Surface-Enhanced Raman Scattering (SERS)

This paper describes the fabrication of gold nanopillar and nanorod arrays and theoretical calculations of electromagnetic fields (EMFs) around ordered arrangements of these nanostructures. The EMFs of both single nanopillars and dimers of nanopillars—having nanoscale gaps between the two adjacent nanopillars forming the dimers—are simulated in this work by employing the finite-difference time-domain method. In the case of simulations for dimers of nanopillars, the nanoscale gaps between the nanopillars are varied between 5 and 20 nm, and calculations of the electromagnetic fields in the vicinity of the nanopillars and in the gaps between the nanopillars were carried out. Fabrication of gold nanopillars in a controlled manner for forming SERS substrates involves focused ion beam (FIB) milling. The nanostructures were fabricated on gold-coated silica, mica, and quartz planar substrates as well as on gold-coated tips of four mode and multimode silica optical fibers.     

Thirty years of resistance: Zig-zag through the plant immune system

Understanding the plant immune system is crucial for using genetics to protect crops from diseases. Plants resist pathogens via a two-tiered innate immune detection-and-response system. The first plant Resistance (R) gene was cloned in 1992 . Since then, many cell-surface pattern recognition receptors (PRRs) have been identified, and R genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) have been cloned. Here, we provide a list of characterized PRRs and NLRs. In addition to immune receptors, many components of immune signaling networks were discovered over the last 30 years. We review the signaling pathways, physiological responses, and molecular regulation of both PRR- and NLR-mediated immunity. Recent studies have reinforced the importance of interactions between the two immune systems. We provide an overview of interactions between PRR- and NLR-mediated immunity, highlighting challenges and perspectives for future research.

A review of major research advances in plant immunity during the last three decades and individual characterized immune receptors, their immune signaling pathways, and interactions between immune systems     

Electromagnetic cellular interactions

Chemical and electrical interaction within and between cells is well established. Just the opposite is true about cellular interactions via other physical fields. The most probable candidate for an other form of cellular interaction is the electromagnetic field. We review theories and experiments on how cells can generate and detect electromagnetic fields generally, and if the cell-generated electromagnetic field can mediate cellular interactions. We do not limit here ourselves to specialized electro-excitable cells. Rather we describe physical processes that are of a more general nature and probably present in almost every type of living cell. The spectral range included is broad; from kHz to the visible part of the electromagnetic spectrum. We show that there is a rather large number of theories on how cells can generate and detect electromagnetic fields and discuss experimental evidence on electromagnetic cellular interactions in the modern scientific literature. Although small, it is continuously accumulating.     

Cell-to-cell signaling through light: just a ghost of chance?

Despite the large number of reports attributing the signaling between detached cell cultures to the electromagnetic phenomena, almost no report so far included a rigorous analysis of the possibility of such signaling.In this paper, we examine the physical feasibility of the electromagnetic communication between cells, especially through light, with regard to the ambient noise illumination. We compare theoretically attainable parameters of communication with experimentally obtained data of the photon emission from cells without a specially pronounced ability of bioluminescence.We show that the weak intensity of the emission together with an unfavorable signal-to-noise ratio, which is typical for natural conditions, represent an important obstacle to the signal detection by cells.     

Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements.     

From Water and Ions to Crowded Biomacromolecules: In Vivo Structuring of a Prokaryotic Cell

Summary: The interactions and processes which structure prokaryotic cytoplasm (water, ions, metabolites, and biomacromolecules) and ensure the fidelity of the cell cycle are reviewed from a physicochemical perspective. Recent spectroscopic and biological evidence shows that water has no active structuring role in the cytoplasm, an unnecessary notion still entertained in the literature; water acts only as a normal solvent and biochemical reactant. Subcellular structuring arises from localizations and interactions of biomacromolecules and from the growth and modifications of their surfaces by catalytic reactions. Biomacromolecular crowding is a fundamental physicochemical characteristic of cells in vivo. Though some biochemical and physiological effects of crowding (excluded volume effect) have been documented, crowding assays with polyglycols, dextrans, etc., do not properly mimic the compositional variety of biomacromolecules in vivo. In vitro crowding assays are now being designed with proteins, which better reflect biomacromolecular environments in vivo, allowing for hydrophobic bonding and screened electrostatic interactions. I elaborate further the concept of complex vectorial biochemistry, where crowded biomacromolecules structure the cytosol into electrolyte pathways and nanopools that electrochemically “wire” the cell. Noncovalent attractions between biomacromolecules transiently supercrowd biomacromolecules into vectorial, semiconducting multiplexes with a high (35 to 95%)-volume fraction of biomacromolecules; consequently, reservoirs of less crowded cytosol appear in order to maintain the experimental average crowding of ∼25% volume fraction. This nonuniform crowding model allows for fast diffusion of biomacromolecules in the uncrowded cytosolic reservoirs, while the supercrowded vectorial multiplexes conserve the remarkable repeatability of the cell cycle by preventing confusing cross talk of concurrent biochemical reactions.

Are the smallest particles of living matter which still exhibit all its functions of the order of magnitude of molecules and atoms, or are they of different order? The first step toward an answer to this question was accomplished by Moritz Nussbaum, who found that if an Infusorian be divided into two pieces, one with and one without a nucleus, only the [former] will continue to live and perform all the functions of self-preservation and development which are characteristic of living organisms. This shows that not only more than two definite substances, but two different structural elements, are needed for life.—Jacques Loeb, 1906 (122)

     

Radiofrequency radiation (900 MHz) induces Egr-1 gene expression and affects cell-cycle control in human neuroblastoma cells

Many environmental signals, including ionizing radiation and UV rays, induce activation of Egr-1 gene, thus affecting cell growth and apoptosis. The paucity and the controversial knowledge about the effect of electromagnetic fields (EMF) exposure of nerve cells prompted us to investigate the bioeffects of radiofrequency (RF) radiation on SH-SY5Y neuroblastoma cells. The effect of a modulated RF field of 900 MHz, generated by a wire patch cell (WPC) antenna exposure system on Egr-1 gene expression, was studied as a function of time. Short-term exposures induced a transient increase in Egr-1 mRNA level paralleled with activation of the MAPK subtypes ERK1/2 and SAPK/JNK. The effects of RF radiations on cell growth rate and apoptosis were also studied. Exposure to RF radiation had an anti-proliferative activity in SH-SY5Y cells with a significant effect observed at 24 h. RF radiation impaired cell cycle progression, reaching a significant G2-M arrest. In addition, the appearance of the sub-G1 peak, a hallmark of apoptosis, was highlighted after a 24-h exposure, together with a significant decrease in mRNA levels of Bcl-2 and survivin genes, both interfering with signaling between G2-M arrest and apoptosis. Our results provide evidence that exposure to a 900 MHz-modulated RF radiation affect both Egr-1 gene expression and cell regulatory functions, involving apoptosis inhibitors like Bcl-2 and survivin, thus providing important insights into a potentially broad mechanism for controlling in vitro cell viability.     

Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements.     

Regulatory role for SRC and phosphatidylinositol 3-kinase in initiation of fibronectin matrix assembly

Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell surface integrin receptors. These interactions activate many intracellular signaling pathways that regulate processes such as cell adhesion, migration, and survival. Here we demonstrate that cells lacking Src family kinases showed reduced ability to assemble FN fibrils as detected by immunofluorescence and by analysis of detergent extracts. The amount of FN matrix was further reduced by treatment with the phosphatidylinositol 3 (PI 3-kinase) inhibitor, wortmannin. CHOalpha5 cells, which are dependent on exogenous FN to initiate fibril formation, also showed significant reductions in matrix when treated with inhibitors of Src and PI 3-kinase. Combination of both inhibitors showed an additive inhibitory effect on assembly, which was concomitant with a loss of focal adhesion kinase phosphorylation. Decreased binding of the 70-kDa amino-terminal FN fragment at matrix assembly sites further supports a role for these kinases early during the process. We propose that these two signaling molecules, which lie downstream of integrins and focal adhesion kinase, are essential for efficient initiation of FN matrix assembly.     

Exposure to GSM RF Fields Does Not Affect Calcium Homeostasis in Human Endothelial Cells,Rat Pheocromocytoma Cells or Rat Hippocampal Neurons

In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an “electromagnetic smog”, with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca²⁺) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca²⁺ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012–2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca²⁺, in addition to Ca²⁺ signals evoked by an InsP₃-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca²⁺ store emptying and store-operated Ca²⁺ entry following application of the Ca²⁺ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca²⁺ signal, recovery rates) were analysed to explore potential impact of radiofrequency field exposure on Ca²⁺ signals. Our data indicate that 900 MHz GSM fields do not affect either basal Ca²⁺ homeostasis or provoked Ca²⁺ signals. Even at the highest field strengths applied, which exceed typical phone exposure levels, we did not observe any changes in cellular Ca²⁺ signals. We conclude that under the conditions employed in our experiments, and using a highly-sensitive assay, we could not detect any consequence of RF exposure.     

Protein interaction data set highlighted with human Ras-MAPK/PI3K signaling pathways

The Ras-MAPK and PI3K-AKT pathways are conserved in metazoan organisms, which involve a series of signaling cascades and form the basis for numerous physiological and pathological processes. Here we report on yeast two hybrid screening results of a protein interaction network around the known components of human Ras-MAPK/PI3K pathways. A total of 42 independent cDNA library screenings resulted in 200 protein-protein interaction (PPI) pairs among 180 molecules. Most of the proteins formed a large cluster that contains 193 PPIs between 169 proteins. Seventy-four interactions indicate high-confidence according to bioinformatics analysis. The prey list contains high enrichment genes with specific Gene Ontology (GO) terms such as response to stress and response to external stimulus. Most interactions link the Ras signaling pathway with various cellular processes. Five interactions were validated by coimmunoprecipitation and colocalization assays in mammalian cells to confirm their in vivo interactions. This protein interaction network provides further insights into the molecular mechanism of Ras-MAPK/PI3K signaling pathways.     

Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2

Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.     

‘Bio-nano interactions: new tools,insights and impacts’: summary of the Royal Society discussion meeting

Bio-nano interactions can be defined as the study of interactions between nanoscale entities and biological systems such as, but not limited to, peptides, proteins, lipids, DNA and other biomolecules, cells and cellular receptors and organisms including humans. Studying bio-nano interactions is particularly useful for understanding engineered materials that have at least one dimension in the nanoscale. Such materials may consist of discrete particles or nanostructured surfaces. Much of biology functions at the nanoscale; therefore, our ability to manipulate materials such that they are taken up at the nanoscale, and engage biological machinery in a designed and purposeful manner, opens new vistas for more efficient diagnostics, therapeutics (treatments) and tissue regeneration, so-called nanomedicine. Additionally, this ability of nanomaterials to interact with and be taken up by cells allows nanomaterials to be used as probes and tools to advance our understanding of cellular functioning. Yet, as a new technology, assessment of the safety of nanomaterials, and the applicability of existing regulatory frameworks for nanomaterials must be investigated in parallel with development of novel applications. The Royal Society meeting ‘Bio-nano interactions: new tools, insights and impacts'' provided an important platform for open dialogue on the current state of knowledge on these issues, bringing together scientists, industry, regulatory and legal experts to concretize existing discourse in science law and policy. This paper summarizes these discussions and the insights that emerged.