Single-cell Fluorescence Microscopy Research Articles

Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing “periplasmic bubbles.” Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2–20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.

The physical boundaries of public goods cooperation between surface-attached bacterial cells.

Bacteria secrete a variety of compounds important for nutrient scavenging, competition mediation and infection establishment. While there is a general consensus that secreted compounds can be shared and therefore have social consequences for the bacterial collective, we know little about the physical limits of such bacterial social interactions. Here, we address this issue by studying the sharing of iron-scavenging siderophores between surface-attached microcolonies of the bacterium Pseudomonas aeruginosa Using single-cell fluorescence microscopy, we show that siderophores, secreted by producers, quickly reach non-producers within a range of 100 µm, and significantly boost their fitness. Producers in turn respond to variation in sharing efficiency by adjusting their pyoverdine investment levels. These social effects wane with larger cell-to-cell distances and on hard surfaces. Thus, our findings reveal the boundaries of compound sharing, and show that sharing is particularly relevant between nearby yet physically separated bacteria on soft surfaces, matching realistic natural conditions such as those encountered in soft tissue infections.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities.

Because of cell to cell variation, it is difficult to obtain statistically significant data on the frequency of exocytosis events (Rexocytosis, t-1 m-2) with traditional single cell electrophysiological or fluorescence microscopy based methods. Here we take the first steps toward a rapid cost-effective surface plasmon resonance (SPR) based method for measuring the Rexocytosis for populations of PC12 cells. The conditions for culturing confluent monolayers on the sensor slides were optimized, and neurotransmitter exocytosis was evoked by injecting solutions with elevated [K+]. Exocytosis caused a shift of the resonance angle (Δθr) that was linearly proportional to Rexocytosis. The Δθr was mainly due to elevated concentration of secretory vesicles close to the cell membrane. The increased vesicle concentration thus acted as a proxy for the Rexocytosis that could not be measured directly. The Δθr was calibrated for Rexocytosis using single cell amperometry on parallel cell cultures. The cell populations were large enough for variation in responses between sensor slides to only reflect actual differences in biological condition. The applicability for drug screening is demonstrated by studying the effects of EGTA, reserpine, and prolonged stimulation by K+.

In vitro neurotoxic hazard characterisation of dinitrophenolic herbicides.

Dinitrophenolic compounds are powerful toxicants with a long history of use in agriculture and industry. While (high) human exposure levels are not uncommon, in particular for agricultural workers during the spraying season, the neurotoxic mechanism(s) that underlie the human health effects are largely unknown. We therefore investigated the in vitro effects of two dinitrophenolic herbicides (DNOC and dinoseb) on a battery of neurotoxicity endpoints in (dopaminergic) rat PC12 cells. Cell viability, mitochondrial activity, oxidative stress and caspase activation were assessed using fluorescence-based bioassays (CFDA, alamar Blue, H2DCFDA and Ac-DEVD-AMC, respectively), whereas changes in intracellular [Ca(2+)]i were assessed using single-cell fluorescence microscopy with Fura-2AM. The combined results demonstrate that exposure to both DNOC and dinoseb is linked to calcium release from the endoplasmic reticulum and activation of caspase-mediated apoptotic pathways. In subsequent experiments, immunofluorescent labelling with specific antibodies was used to determine changes in intracellular α-synuclein levels, demonstrating that both DNOC and dinoseb increase levels of intracellular α-synuclein. The combined results indicate that in vitro exposure to DNOC and dinoseb activates pathways that are not only involved in acute neurotoxicity but also in long-term effects as seen in neurodegeneration.

CRISPR-mediated control of the bacterial initiation of replication.

Programmable control of the cell cycle has been shown to be a powerful tool in cell-biology studies. Here, we develop a novel system for controlling the bacterial cell cycle, based on binding of CRISPR/dCas9 to the origin-of-replication locus. Initiation of replication of bacterial chromosomes is accurately regulated by the DnaA protein, which promotes the unwinding of DNA at oriC. We demonstrate that the binding of CRISPR/dCas9 to any position within origin or replication blocks the initiation of replication. Serial-dilution plating, single-cell fluorescence microscopy, and flow-cytometry experiments show that ongoing rounds of chromosome replication are finished upon CRISPR/dCas9 binding, but no new rounds are initiated. Upon arrest, cells stay metabolically active and accumulate cell mass. We find that elevating the temperature from 37 to 42°C releases the CRISR/dCas9 replication inhibition, and we use this feature to recover cells from the arrest. Our simple and robust method of controlling the bacterial cell cycle is a useful asset for synthetic biology and DNA-replication studies in particular. The inactivation of CRISPR/dCas9 binding at elevated temperatures may furthermore be of wide interest for CRISPR/Cas9 applications in genomic engineering.

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions.

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult.

Accurate concentration control of mitochondria and nucleoids.

All cellular materials are partitioned between daughters at cell division, but by various mechanisms and with different accuracy. In the yeast Schizosaccharomyces pombe, the mitochondria are pushed to the cell poles by the spindle. We found that mitochondria spatially reequilibrate just before division, and that the mitochondrial volume and DNA-containing nucleoids instead segregate in proportion to the cytoplasm inherited by each daughter. However, nucleoid partitioning errors are suppressed by control at two levels: Mitochondrial volume is actively distributed throughout a cell, and nucleoids are spaced out in semiregular arrays within mitochondria. During the cell cycle, both mitochondria and nucleoids appear to be produced without feedback, creating a net control of fluctuations that is just accurate enough to avoid substantial growth defects.

Mitochondria migration during mitosis

Cell Biology Every time a cell divides, it is faced with the problem of ensuring adequate distribution of all components to the daughter cells. This includes components that are present in relatively small numbers, like the mitochondria and their DNA-containing nucleoids. Jajoo et al. used quantitative time-lapse fluorescence microscopy of single yeast cells to explore how this works. Unlike chromosomes or macromolecules, mitochondria and nucleoids are distributed in proportion to the volume of cytoplasm received by each daughter cell. Partitioning errors are kept low by even distribution of the mitochondrial volume and roughly equal spacing of the nucleoids before they are distributed. Science , this issue p. [169][1] [1]: /lookup/doi/10.1126/science.aaa8714

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

Background/Aims: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca²⁺ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca²⁺ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca²⁺ uptake upon intracellular Ca²⁺ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca²⁺ overload and subsequent cancer cell death.

In Vitro Developmental Neurotoxicity Following Chronic Exposure to 50 Hz Extremely Low-Frequency Electromagnetic Fields in Primary Rat Cortical Cultures.

Exposure to 50-60 Hz extremely low-frequency electromagnetic fields (ELF-EMFs) has increased considerably over the last decades. Several epidemiological studies suggested that ELF-EMF exposure is associated with adverse health effects, including neurotoxicity. However, these studies are debated as results are often contradictory and the possible underlying mechanisms are unknown. Since the developing nervous system is particularly vulnerable to insults, we investigate effects of chronic, developmental ELF-EMF exposure invitro. Primary rat cortical neurons received 7 days developmental exposure to 50 Hz block-pulsed ELF-EMF (0-1000 μT) to assess effects on cell viability (Alamar Blue/CFDA assay), calcium homeostasis (single cell fluorescence microscopy), neurite outgrowth (β(III)-Tubulin immunofluorescent staining), and spontaneous neuronal activity (multi-electrode arrays). Our data demonstrate that cell viability is not affected by developmental ELF-EMF (0-1000 μT) exposure. Depolarization- and glutamate-evoked increases in intracellular calcium concentration ([Ca(2+)]i) are slightly increased at 1 μT, whereas both basal and stimulation-evoked [Ca(2+)]i show a modest inhibition at 1000 μT. Subsequent morphological analysis indicated that neurite length is unaffected up to 100 μT, but increased at 1000 μT. However, neuronal activity appeared largely unaltered following chronic ELF-EMF exposure up to 1000 μT. The effects of ELF-EMF exposure were small and largely restricted to the highest field strength (1000 μT), ie, 10000 times above background exposure and well above current residential exposure limits. Our combined data therefore indicate that chronic ELF-EMF exposure has only limited (developmental) neurotoxic potential invitro.

Bisnaphthalimidopropyl diaminodicyclohexylmethane induces DNA damage and repair instability in triple negative breast cancer cells via p21 expression

Bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) bisintercalates to DNA and is a potential anti-cancer therapeutic. In an attempt to elucidate the mechanism(s) underlying the potential of BNIPDaCHM; earlier work was extended to investigate its effect on DNA damage and repair as well as cell cycle modulation, in a triple negative breast cancer (TNBC) cell line in vitro.BNIPDaCHM significantly decreased cell viability in a concentration (≥5 μM) and time (≥24 h) dependent manner. The mechanism of this growth inhibition involved alterations to cell cycle progression, an increase in the sub-G1 population and changes to plasma membrane integrity/permeability observed by flow cytometry and fluorescence microscopy with acridine orange/ethidium bromide staining. Using single cell gel electrophoresis (Comet assay) and fluorescence microscopy to detect γ-H2AX-foci expression; it was found that after 4 h, ≥ 0.1 μM BNIPDaCHM treatment-induced significant DNA double strand breaks (DSBs). Moreover, exposure to a non-genotoxic concentration of BNIPDaCHM induced a significant decrease in the repair of oxidative DNA strand breaks induced by hydrogen peroxide. Also, BNIPDaCHM-treatment induced a significant time dependent increase in p21Waf/Cip1 mRNA expression but, did not alter p53 mRNA expression.In conclusion, BNIPDaCHM treatment in MDA-MB-231 cells was associated with a significant induction of DNA DSBs and inhibition of DNA repair at non-genotoxic concentrations via p53-independent expression of p21Waf1/Cip1. The latter may be a consequence of novel interactions between BNIPDaCHM and MDA-MB-231 cells which adds to the spectrum of therapeutically relevant activities that may be exploited in the future design and development of naphthalimide-based therapeutics.

DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear bis(triazacyclonane) copper complex

The dinuclear copper(II) complex [Cu2{bcmp(-H)}(μ-OH)](NO3)2·H2O (1, bcmp=2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol) has been synthesized and characterized by electrospray ionization mass spectrometry, potentiometric titration and cyclovoltammetry. The X-ray structure of the analogous perchlorate salt [Cu2{bcmp(-H)}(μ-OH)](ClO4)2·2.5H2O (2) was determined. Cytotoxicity studies showed very promising activity of 1 against various pancreatic tumor cell lines with IC50 values comparable or even lower than those of cisplatin. The Cu complex displayed low toxicity against a human non-tumor cell line (HEK 293) demonstrating selectivity for cancer cells. 1 converts supercoiled pUC19 plasmid DNA into the nicked form at micromolar concentrations in the absence of added reductants. A detailed kinetic study on the hydrolysis of the DNA model bis(2,4-dinitrophenyl) phosphate (BDNPP) has been performed. 1 hydrolyses BDNPP with a second order rate constant of 0.047Ms−1 at pH8 and 40°C. Finally, single cell electrophoresis (comet assay) and fluorescence microscopy analysis showed that 1 interacts with cellular DNA and induces apoptotic cell death of Capan-1 pancreatic cancer cells. Western blotting analysis indicated that the Cu complex activates the p53 dependent pathway of apoptosis.

Comparison of plate reader-based methods with fluorescence microscopy for measurements of intracellular calcium levels for the assessment of in vitro neurotoxicity

The intracellular calcium concentration ([Ca2+]i) is an important readout for in vitro neurotoxicity since calcium is critically involved in many essential neurobiological processes, including neurotransmission, neurodegeneration and neurodevelopment. [Ca2+]i is often measured with considerable throughput at the level of cell populations with plate reader-based assays or with lower throughput at the level of individual cells with fluorescence microscopy. However, these methodologies yield different quantitative and qualitative results. In recent years, we demonstrated that the resolution and sensitivity of fluorescence microscopy is superior compared to plate reader-based assays. However, it is currently unclear if the use of plate reader-based assays results in more ‘false negatives’ or ‘false positives’ in neurotoxicity screening studies. In the present study, we therefore compared a plate reader-based assay with fluorescence microscopy using a small test set of environmental pollutants consisting of dieldrin, lindane, polychlorinated biphenyl 53 (PCB53) and tetrabromobisphenol-A (TBBPA). Using single-cell fluorescence microscopy, we demonstrate that all test chemicals reduce the depolarization-evoked increase in [Ca2+]i, whereas lindane, PCB53 and TBBPA also increase basal [Ca2+]i, though via different mechanisms. Importantly, none of these effects were confirmed with the plate reader-based assay. We therefore conclude that standard plate reader-based methods are not sufficiently sensitive and reliable to measure the highly dynamic and transient changes in [Ca2+]i that occur during chemical exposure.

Inhibition of voltage-gated calcium channels as common mode of action for (mixtures of) distinct classes of insecticides.

Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78nM, 239nM, 250nM, and 899nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10μM) induced a modest increase in basal [Ca(2+)]i, amounting to 68 ± 8nM and 53 ± 8nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10μM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Because inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.

A set of versatile brick vectors and promoters for the assembly, expression, and integration of synthetic operons in Methylobacterium extorquens AM1 and other alphaproteobacteria.

The discipline of synthetic biology requires standardized tools and genetic elements to construct novel functionalities in microorganisms; yet, many model systems still lack such tools. Here, we describe a novel set of vectors that allows the convenient construction of synthetic operons in Methylobacterium extorquens AM1, an important alphaproteobacterial model organism for methylotrophy and a promising platform organism for methanol-based biotechnology. In addition, we provide a set of constitutive alphaproteobacterial promoters of different strengths that were characterized in detail by two approaches: on the single-cell scale and on the cell population level. Finally, we describe a straightforward strategy to deliver synthetic constructs to the genome of M. extorquens AM1 and other Alphaproteobacteria. This study defines a new standard to systematically characterize genetic parts for their use in M. extorquens AM1 by using single-cell fluorescence microscopy and opens the toolbox for synthetic biological applications in M. extorquens AM1 and other alphaproteobacterial model systems.

Assessment of the neurotoxic potential of exposure to 50 Hz extremely low frequency electromagnetic fields (ELF-EMF) in naïve and chemically stressed PC12 cells

Increasing exposure to extremely low frequency electromagnetic fields (ELF-EMF), generated by power lines and electric appliances, raises concern about potential adverse health effects of ELF-EMF. The central nervous system is expected to be particularly vulnerable to ELF-EMF as its function strongly depends on electrical excitability. We therefore investigated effects of acute (30min) and sub-chronic (48h) exposure to 50Hz ELF-EMF on naïve and chemically stressed pheochromocytoma (PC12) cells. The latter have higher levels of iron and/or reactive oxygen species (ROS) and display increased vulnerability to environmental insults. Effects of ELF-EMF on Ca2+-homeostasis, ROS production and membrane integrity were assessed using Fura-2 single cell fluorescence microscopy, H2-DCFDA and CFDA assays, respectively. Our data demonstrate that acute exposure of naïve PC12 cells to 50Hz ELF-EMF up to 1000μT fails to affect basal or depolarization-evoked [Ca2+]i. Moreover, sub-chronic ELF-EMF exposure up to 1000μT has no consistent effects on Ca2+-homeostasis in naïve PC12 cells and does not affect ROS production and membrane integrity. Notably, in chemically stressed PC12 cells both acute and sub-chronic ELF-EMF exposure also failed to exert consistent effects on Ca2+-homeostasis, ROS production and membrane integrity. Our combined findings thus indicate that exposure to 50Hz ELF-EMF up to 1000μT, i.e. 10,000 times above background exposure, does not induce neurotoxic effects in vitro, neither in naïve nor in chemically stressed PC12 cells. Though our data require confirmation, e.g. in developing neuronal cells in vitro or (developing) animals, it appears that the neurotoxic risk of ELF-EMF exposure is limited.

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

BackgroundHeterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation.ResultsWe describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR.ConclusionsSingle cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.

Acute disturbance of calcium homeostasis in PC12 cells as a novel mechanism of action for (sub)micromolar concentrations of organophosphate insecticides

Organophosphates (OPs) and carbamates are widely used insecticides that exert their neurotoxicity via inhibition of acetylcholine esterase (AChE) and subsequent overexcitation. OPs can induce additional neurotoxic effects at concentrations below those for inhibition of AChE, indicating other mechanisms of action are also involved. Since tight regulation of the intracellular calcium concentration ([Ca2+]i) is essential for proper neuronal development and function, effects of one carbamate (carbaryl) and two OPs (chlorpyrifos, parathion-ethyl) as well as their -oxon metabolites on [Ca2+]i were investigated. Effects of acute (20min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca2+]i were measured in fura-2-loaded PC12 cells using single-cell fluorescence microscopy. Acute exposure to chlorpyrifos and its metabolite chlorpyrifos-oxon (10μM) induced a modest increase in basal [Ca2+]i. More importantly, the tested OPs concentration-dependently inhibited depolarization-evoked [Ca2+]i. Chlorpyrifos already induced a ∼30% inhibition at 0.1μM and a 100% inhibition at 10μM (IC50=0.43μM), whereas parathion-ethyl inhibited the depolarization-evoked [Ca2+]i increase with ∼70% at 10μM. Interestingly, -oxon metabolites were more potent inhibitors of AChE, but were less potent inhibitors of depolarization-evoked [Ca2+]i compared to their parent compound (chlorpyrifos-oxon) or were even without effect (paraoxon-ethyl and -methyl). Similarly, acute exposure to carbaryl had no effect on [Ca2+]i. Exposure to mixtures of chlorpyrifos with its oxon-analog or with parathion-ethyl did not increase the degree of inhibition, indicating additivity does not apply. These data demonstrate that concentration-dependent inhibition of depolarization-evoked [Ca2+]i is a novel mechanism of action of (sub)micromolar concentrations of OPs that could partly underlie OP-induced neurotoxicity.

A Quantitative Narrative for the Life Cycle of Bacteriophage Lambda

The goal of my lab is to form a quantitative narrative for the fundamental processes driving the living cell. This narrative is built upon precise measurements performed in individual cells, at the level of individual molecules and discrete events in space and time. To achieve this level of detail, we are using a synthesis of approaches: classical molecular biology and biochemistry; single-cell and single-molecule fluorescence microscopy; advanced image- and data analysis algorithms. By using simple, coarse-grained theoretical models, we are able to distill our result into general principles, which can then be compared across organisms. I will present a few examples from our work, focusing on the study of the life cycle of bacteriophage lambda, a simple paradigm for cellular decision making.