Can We See PIP 3 and Hydrogen Peroxide with a Single Probe?

Abstract

A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H₂O₂) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP₃) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H₂O₂. The dynamics of PIP₃ and H₂O₂ generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts. Antioxid. Redox Signal. 17, 505–512.

Introduction

F luorescent microscopy is a powerful method to study cell signaling in vivo. A wide spectrum of fluorescent proteins (FPs), FP-based sensors, and small-molecule chemical dyes allow visualization of many intracellular signaling events. Combinations of various fluorophores with distinct excitation and emission spectra in a single cell enable monitoring of multiple cellular processes simultaneously, usually termed multiparameter imaging (see Supplementary Data; Supplementary Data are available online at www.liebertonline.com/ars).

Phosphorylated forms of phosphoinositide lipids (PIPs) transduce signals via recruiting the PIP-binding domains that vary in their selectivity toward the number and position of phosphates of the inositol ring, allowing a fairly precise specificity of downstream signaling activation (8). One important lipid messenger is phosphatidylinositol 3,4,5-trisphosphate (PIP₃). Phosphatidylinositol 3-kinase (PI3K) phosphorylates PI(4,5)P₂ to PIP₃, while the lipid phosphatase and tensin homolog (PTEN) reverses the phosphorylation (7). FPs fused with PIP_n-sensitive protein domains allow monitoring of PIP_n formation by translocation of the fluorescently labeled domain from the cytosol to the plasma membrane (PM) (8).

The global signaling activity of hydrogen peroxide (H₂O₂) is much less commonly addressed (3). H₂O₂ selectively and reversibly oxidizes a small population of cysteines that tend to be deprotonated at physiological pH (9). H₂O₂ production by NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes and generation of PIP₃ by receptor tyrosine kinase activation can be highly cooperative: The NOX subunits p47 and p40 are recruited to membranes by their PX domains that recognize products of PI3K activity and activate the oxidase activity (4). The subsequent H₂O₂ production then generates a positive feedback loop by oxidizing the active-site thiolate of PTEN, leading to an increased lifetime of PI3K products (5). To delineate the interplay of both signaling molecules more precisely, simultaneous detection of both PI3K activity and H₂O₂ levels would be necessary.

Innovation

Combinations of various fluorophores with distinct excitation and emission spectra in a single cell enable monitoring of multiple cellular processes simultaneously, usually termed multiparameter imaging. We successfully combined two different readouts in a single probe: ratiometric for hydrogen peroxide (H₂O₂) and translocation for phosphatidylinositol 3-kinase. The probe simultaneously detects the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP₃) and H₂O₂, showing that the two signals are highly cooperative. The performance of the sensor was tested in two cellular models, including immunological synapse formation of primary human T-cells, where we use it for kinetical measurements of two second messengers simultaneously. Making use of the subcellular localization of PIP₃ at the immune synapse, we are able to analyze H₂O₂ in subcellular domains with good resolution and a very good signal-to-noise ratio. Our data show as a proof of principle that different readouts can be easily combined in a single sensor, increasing a number of measured substances in a multiparameter imaging and minimizing a number of expression constructs.

Genetically encoded sensor for H₂O₂, HyPer, is based on insertion of a yellow FP into regulatory domain of Escherichia coli H₂O₂ sensor OxyR (2). Upon oxidation and S-S bond formation within the OxyR domain, the excitation spectrum of HyPer changes in a ratiometric manner, proportionally decreasing its 420 nm and increasing its 500 nm excitation peak. Considering the interdependence of H₂O₂ and PI3K signaling, the simultaneous and spatially resolved measurement of both is desired in many cases. For this purpose, we have generated a sensor that reported changes in both PIP₃ and H₂O₂ concentration utilizing two different types of readout. Fusing HyPer with a PIP₃-sensitive PH domain should allow visualization of both PIP₃ generation (by translocation of the probe from the cytoplasm to the PM) and of H₂O₂ generation (by monitoring the excitation ratio 500/420 nm of HyPer).

Results, Discussion, and Future Directions

To generate a dual, PI3K and H₂O₂, sensor, we fused HyPer with a mutated PH domain (E41K) of Bruton's tyrosine kinase (BTK). The resulting reporter, named PIP-SHOW (PIP₃ and − SH Oxidation Watching) (Fig. 1A), emitted fluorescence signals in the expected wavelength range (500–550 nm) when expressed in NIH-3T3 fibroblasts. We then verified if the PIP-SHOW retained the ability of both domains, BTK-PH-E41K and HyPer, to respond to the respective stimuli, H₂O₂ and PIP₃. Addition of H₂O₂ resulted in the expected change in the probe's excitation ratio (Fig. 1B) similar to that of HyPer. Incubation of cells with 10 μM of a membrane-permeant photoactivatable version of PIP₃ (cgPIP₃/AM) and subsequent brief illumination with 405 nm light led to partial redistribution of the probe to the PM (Fig. 1C). Hence, both the translocation domain and the HyPer performed correctly.

FIG. 1.

PIP-SHOW reports changes in PIP₃ and H₂O₂ levels by two different types of readouts: translocation of the probe and excitation peaks ratio change, respectively. (A) PIP-SHOW consists of two parts, the PIP₃-sensitive PH domain of BTK and the H₂O₂ sensor HyPer. An E41K point mutation was introduced into the PH domain to increase sensitivity. (B) External H₂O₂ (50 μM) causes change in the sensor excitation ratio, F500/F420, in the cytoplasm of HeLa-Kyoto cell. (C) Photouncaging of synthetic PIP₃ causes redistribution of the probe to the PM domains. Arrows indicate zones of elevated PIP₃. Scale bar=10 μm. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars). H₂O₂, hydrogen peroxide; PIP, phosphatidylinositol phosphate; PIP₃, phosphatidylinositol 3,4,5-trisphosphate; PM, plasma membrane; BTK, Bruton's tyrosine kinase.

To further characterize PIP-SHOW, we stimulated NIH-3T3 fibroblasts with platelet-derived growth factor (PDGF) and analyzed the fluorescence signals over time. Addition of PDGF initiated translocation of the probe to the PM reflecting PI3K activation and caused a significant change in the excitation ratio 500/420 nm, indicating generation of H₂O₂ (Fig. 2A; Supplementary Video S1). To quantify PI3K activity and H₂O₂ production in more detail, we analyzed their respective dynamics within single cells. Both signals were initially highly cooperative (Fig. 2B–E). Furthermore, Figure 2A–E showed that while both signals rose at similar times, PI3K accumulation reversed with a much slower time course, while H₂O₂ production was strictly transient. This indicates that both signals are not necessarily coupled for prolonged periods of time and highlights the importance of a dual sensor such as PIP-SHOW. This difference in kinetics could result from different sensitivities of the two domains comprising PIP-SHOW. However, this scenario is unlikely given their high sensitivity: BTK-PH-E41K domain was able to detect resting PIP₃ in the membrane of nonstimulated T-cells (see below) and the OxyR-RD domain of HyPer has extremely high reaction rates with H₂O₂ (10⁷ M⁻¹s⁻¹), enabling intracellular H₂O₂ detection even in the presence of peroxiredoxins and glutathione peroxidases. PIP-SHOW does not completely translocate to the PM, and this allows comparison of the excitation ratio between the PM and the cytoplasm. Figure 2A shows that NIH-3T3 cells generate H₂O₂ in the vicinity of the PM, and that this H₂O₂ is unable to distribute uniformly throughout the cytoplasm. Because PI3K activity acts upstream of NOX/DUOX activation (4), we analyzed the effect of the PI3K inhibitor wortmannin on PI3K activity and H₂O₂ production, after stimulation with PDGF. Figure 2F shows that wortmannin prevented PI3K translocation but also inhibited H₂O₂ production. To further characterize PIP-SHOW, we inhibited NOX enzymes with diphenyleniodonium (DPI). Obviously, DPI led to a rapid drop in PIP-SHOW ratio reflecting decrease in H₂O₂ production (Fig. 2G). Because we did not observe a decrease in PIP₃ levels after DPI treatment, a potentially possible feedback loop between PI3K and NOX/DUOX activity via PTEN inhibition can be excluded.

FIG. 2.

Imaging PIP₃ and H₂O₂ with PIP-SHOW in NIH-3T3 cells stimulated with PDGF. (A) Widefield fluorescence images of PIP-SHOW–expressing NIH-3T3 cells at indicated time points (in minutes) after stimulation of the cells with 10 ng/ml PDGF. Upper row of images represent subcellular distribution of PIP-SHOW ratio reflecting changes in H₂O₂ level. Middle and lower rows of images show subcellular distribution of PIP-SHOW and changes in fluorescence intensity in each imaging channel. Individual cells are highlighted on the upper left corner ratio panel and numbered 1 to 4. Scale bar=15 μm. The panel is representative of 20 cells from three experiments. (B–E) Time course of PI3K activation and H₂O₂ production by the cells 1 to 4 shown on (A). Black lines reflect redistribution of PIP-SHOW to PM, red lines reflect H₂O₂ level changes in the cells. (F) Pretreatment of the NIH-3T3 cells with wortmannin (500 nM) abolished elevation of both PIP₃ and H₂O₂. Panel represents a typical time course of PIP₃ and H₂O₂ production by the cells pretreated with wortmannin. (G) The NOX inhibitor DPI (10 μM) inhibits H₂O₂ production but does not lead to a drop in PIP₃ levels. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars). PI3K, phosphatidylinositol 3-kinase; PDGF, platelet-derived growth factor; DPI, diphenyleniodonium; NOX, NADPH oxidase.

Next, we utilized PIP-SHOW to study lipid and redox signaling events in the first phase of CD4⁺human T helper (T_H) cell activation (Supplementary Data). Transient expression of PIP-SHOW in human T_H cells led to its localization within the cytoplasm as well as within the nucleus. However, a fraction of the probe showed membrane localization, indicating of pre-existing PI3K activity (Fig. 3A). Stimulation of T_H cells expressing PIP-SHOW with anti-CD3/CD28-coated beads led to establishment of stable contacts, immunological synapses (IS), between T_H cells and the beads (Fig. 3A; Supplementary Video S2). This was followed by a massive and immediate redistribution of PIP-SHOW to the IS indicative of locally elevated PIP₃ levels. The probe remained in the contact region for the duration of the experiment (up to 1 h). Notably, PI3K activity decreased at the rest of the PM of the activated cell as the ring-like fluorescence pattern disappeared immediately after IS formation (Fig. 3A). Furthermore, IS formation also led to a rapid elevation of H₂O₂, ∼1 min after translocation of the PIP₃ sensor (Fig. 3; Supplementary Video S2). Interestingly, in most of the cells H₂O₂ production was highest in the region adjacent to the central synapse (Fig. 3E), implicating a ring-like localization of NOX/DUOX enzymes around the central IS. DPI quickly attenuated H₂O₂ production (Fig. 3F), suggesting that PM-localized NOX/DUOX are the source of H₂O₂. Similar to NIH-3T3 cells, there was no fast positive feedback loop between PI3K and NOX/DUOX activities, as we did not observe a decrease in PIP₃ upon addition of DPI (Fig. 3F). However, it is possible that the oxidant-induced decrease in phosphatase activity is only slowly reversible and acts in a long-term memory-like fashion. This might be the reason that a decrease in PIP₃ concentration was not detected in the time course of this experiment. Inhibition of PI3K by wortmannin, however, led to a rapid decrease in both PI3K activity and H₂O₂ production (Fig. 3G) as expected.

FIG. 3.

PIP-SHOW detects PI3K activity and H₂O₂ production in the human T_H cell upon IS formation. (A) Widefield fluorescence images of T_H cell forming a contact with anti-CD3/CD28–coated beads (added at timepoint zero). Numbers indicate time points in minutes. Upper row of the images represents transmitted light channel (beads are dense black); two next lower rows show distribution of the fluorescence sequentially excited at 420 and 505 nm; lowest row represents distribution of H₂O₂ represented by the 505/420 excitation ratio. Scale bar=10 μm. (B, C) Time course of PI3K activation and H₂O₂ production in individual T_H cells. Black line reflects redistribution of PIP-SHOW to the IS; red line reflects H₂O₂ level changes in the cell. (D) Mean values of PI3K activity and H₂O₂ production in 28 T_H cells from three experiments and two donors. Error bars±standard error of mean. (E) Distribution of H₂O₂ between the IS and the cytoplasm in 44 cells from four experiments expressed as the ratio of the HyPer signal in the two subcompartments. Equal H₂O₂ concentration at the IS and in the cytoplasm is indicated by a ratio 1. (F) NOX inhibitor DPI (10 μM) reduces H₂O₂ production in the human T_H cell upon IS formation. (G) PI3K inhibitor wortmannin (500 nM) leads to a decrease in both PIP₃ and H₂O₂ levels. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars). IS, immunological synapse; T_H, T helper; a.u., arbitrary units.

We demonstrated that the novel dual-parameter sensor PIP-SHOW is well suited for simultaneous monitoring of PIP₃ and H₂O₂ levels and can thereby serve as a prototype for indicators with combined readouts. The concept of a translocating domain fused to a ratiometric sensor is widely applicable to other combinations of intracellular signaling parameters as well. The second component of PIP-SHOW, HyPer, was designed and used to report H₂O₂ levels (2). Because HyPer is a protein of moderate size (∼2 green fluorescent protein [GFP] molecules), it can be fused with subcellular localization tags. As HyPer is derived from a bacterially encoded protein, it is unlikely to interfere with other proteins and signaling pathways in mammalian cells. An important advantage of HyPer and therefore PIP-SHOW is the ratiometric response to H₂O₂, which is independent on the relative amount of the sensor, its redistribution, cell movement, or shape change. Indeed, in the theoretical case of intensiometric readout for H₂O₂, it would be impossible to discriminate between PIP₃ and near-membrane H₂O₂ during PI3K activation and PIP-SHOW translocation. Thus, the ratiometric nature of HyPer is necessary to allow the simultaneous quantification of H₂O₂ during PI3K activity. In addition, we also showed that PIP-SHOW is combinable with RFP-based sensors, allowing synchronous measurements of an additional third parameter (Supplementary Fig. S1; Supplementary Data).

We validated PIP-SHOW using two cellular systems in which both PI3K and NOX/DUOX (reactive oxygen species [ROS]) were already shown to be important determinants of several signaling pathways. Our results provide relevant information about the effectiveness of the sensor but also new information about the cooperative dynamics of the two signaling systems. While PDGF stimulated fibroblasts are a well-documented experimental model to study lipid and redox signaling, not much was known about the role of ROS and their interaction with PI3K and, respectively, PIP₃ in human T_H cells.

In both cellular systems, H₂O₂ and PIP₃ demonstrated a high degree of synchronicity, particularly in the initial phase after stimulation (see Figs. 2 and 3). In fibroblasts, however, the decrease in H₂O₂ was accompanied by decrease in PIP₃ only in a fraction of the cells. In polarized T cells, on the other hand, the PI3K activity rapidly redistributed and increased at the IS and decreased in the rest of the PM. The H₂O₂ distribution demonstrated a higher degree of heterogeneity: whereas in most of the cells highest H₂O₂ levels were detected at the IS, in some H₂O₂ was more evenly distributed (Fig. 3E). Nevertheless, in most cases the source of H₂O₂ was visibly associated with either the PM or, more specifically, the IS. Interestingly, in contrast to fibroblasts, PI3K activity in the stimulated T_H cells started before H₂O₂ production was detectable with HyPer (Figs. 2 and 3). More physiological studies are needed to determine the functional role of local ROS microdomains close to the IS not only in T_H but also in other immune cells.

PIP₃ concentration at the PM is a result of the concerted action of the PI3-kinases and lipid phosphatases. The latter contain, similar to protein tyrosine phosphatases, thiolates in the active site, rendering them redox sensitive. H₂O₂ was shown to oxidize the lipid phosphatase PTEN, enabling positive feedback loop in the phosphorylation cascade. We would therefore expect to see a decrease in PIP₃ concentration upon elimination of H₂O₂. The kinetics of PIP-SHOW disulphide reduction should be similar to the reduction of the phosphatases. To our surprise, in both experimental models we did not see an effect of NOX inhibition on the PIP₃ content. While the HyPer signal (H₂O₂) decreased rapidly upon addition of DPI, the localization of the PH domain did not change, indicating that PIP₃ concentrations did not decrease significantly. A possible explanation is that in strongly stimulated cells, PI3K activity is dominating over phosphatases activity in such a way that inhibition of the phosphatases (at least lipid phosphatases) by H₂O₂ does not shift the equilibrium significantly. Another interpretation might be an increased sensitivity of the probe to PIP₃ due to the E41K mutation that prevents retrograde translocation of the sensor. However, this explanation appears unlikely because inhibition of PI3K by wortmannin led to rapid redistribution of the probe from PM to cytoplasm, implicating a high lipid phosphatase activity.

In summary, we successfully combined two different readouts in a single probe. In fact, a variety of translocation-based sensor domains are suitable to be fused with any ratiometric indicator regardless whether it is single fluorophore or a Forster resonance energy transfer pair. Ratiometric sensors can serve as the fluorescence tags instead of conventional FPs, and they can provide additional read-out parameters, such as 2nd messenger concentrations in time-lapse imaging experiments. Obvious other benefits are that only a small part of the usable spectra is occupied and additional colors may be used for other sensors in a multiparameter imaging setup. Most importantly, only one expression construct is needed for the dual-parameter readout, which makes the relative quantification and the interpretation of the results much easier.

Notes

Materials used

H₂O₂, DPI, EGF, and PDGF-BB were purchased from Sigma. Dulbecco-phosphate-buffered saline, Dulbecco's modified Eagle's medium (DMEM), Opti–minimal essential medium (MEM), MEM, fetal calf serum (FCS), and FuGene6 transfection reagent were from Invitrogen. Glass-bottomed dishes were fromMatTec. NIH-3T3 cells were from ATCC. HeLa-Kyoto cell line was provided by EMBL. Encyclo polymerase chain reaction (PCR) kit and HyPer expression vectors were from Evrogen. Restriction endonucleaseswere from SibEnzyme.

DNA constructs

To make PIP-SHOW, the PH-domain coding region of BTK was amplified from pEGFP-BtkPH encoding vector using the primers 5′-ATCCGCTAGCATGGCCGCAGTGATTCTG GAGA-3′ and 5′-CGGTGGATCCCCGTTCTCCAAAATTT GGCAGCCCA-3′. The PCR product was digested with NheI and BamHI and cloned into pHyPer-dMito vector (Evrogen) in place of double MTS-coding sequence. It has been shown that E41K mutation increases the affinity of the BTK-PH to PIP₃ (1). To introduce E41K mutation, site-directed mutagenesis of Btk PH-domain was applied using the PCR overlap extension procedure with the primers 5′-CTCCTACTATA AGTATGACTTTGA-3′ and 5′-TCAAAGTCATACTTATAG TAGGAG-3′. All constructs were confirmed by sequence analysis.

Cell culture and transfection

NIH-3T3 cells were cultured in DMEM supplemented with 10% FCS at 37°C in an atmosphere containing 95% air and 5% CO₂. Cells were split every 2nd day and seeded on glass bottom dishes. Twenty-four hours later cells were transfected by the mixture of vector DNA and FuGene6 transfection reagent according to the manufacturer recommendations.

Human T_H cells were isolated from leukocyte reduction filters from healthy blood donors. First, peripheral blood lymphocytes (PBLs) were purified by a density gradient centrifugation at 450 g for 30 min at room temperature (Ficoll-Paque PLUS, Amersham Biosciences), while the remaining red blood cells were removed using a lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA [pH 7.3]). PBLs were resuspended in PBS buffer containing 0.5% bovine serum albumin, and CD4⁺ T_H cells were negatively isolated using CD4⁺ negative isolation kit (Invitrogen). Naïve T_H cells were transfected ∼ 4 h after isolation using Nucleofector II electroporator (Lonza, program U-014) and Human T cell Nucleofector kit (Lonza) according to manufacturer's instructions. Per one million cells 1 μg DNA was electroporated 24 to 48 h after transfection, and the cells were attached (∼10 min, 37°C) to poly-l-ornithine–coated (0.1 mg/ml) glass coverslips, and imaged.

Imaging

The T_h cells were imaged at 37°C using a Zeiss Cell Observer HS widefield microscope equipped with a 40× Fluar oil lense (N.A. 1.3), an LED fluorescence lamp (Colibri, Zeiss), an and a photometric evolve: 512 EMCCD camera at binning 1×1. Filters were a CFP, YFP dualband pinkel set (55HE, Zeiss). Fluorescence was recorded using excitation at 420 and 505 nm and emission at 515 nm. NIH-3T3 cells were imaged using Leica 6000 widefield microscope equipped with an HCX PL APO lbd.BL 63×1.4NA oil objective and an environmental chamber. Fluorescence was excited sequentially via 427/10 and 504/12 band-pass excitation filters. Emission of the probe was collected every 10 or 30 s using a 525/50 band-pass emission filter. After three to five images were acquired, 10 ng/ml PDGF was added.

cgPI(3,4,5)P₃/AM treatment and uncaging

Caged, membrane permeable form of PI(3,4,5)P₃, cgPI(3,4,5)P₃/AM (6) was dissolved in dimethyl sulfoxide (DMSO) at stock concentration of 10 mM. Just before applying to cells it was mixed with 10% pluronic/DMSO solution (Invitrogen) in 1:1 ratio to facilitate cell entry. cgPI(3,4,5)P₃/AM was used on cells in a concentration of 10 μM. Uncaging and imaging were performed on Carl Zeiss LSM 510-META confocal microscope, equipped with environment control box (37°C, 0% CO₂), using HCX PL APO Ibd. 60×1.4NA oil objective. Cells were preincubated with cgPI(3,4,5)P₃/AM 2 h before uncaging in the imaging medium. For uncaging, cells were illuminated with 405 nm laser (7%) every 10 s. Imaging of PIP₃-SHOW was done using 488 nm laser excitation (5%) with 10 s time resolution.

Time series processing

Time series were analyzed using ImageJ software. For H₂O₂ dynamics calculation, stacks corresponding to 420 and 500 nm excitation peaks of PIP-SHOW were converted to 32 bit after background subtraction. 420-nm stack was thresholded to remove pixel values from background (Not-a-Number function). A 500-nm stack was divided by the corresponding 420-nm stack frame by frame. The resulting stack was depicted in pseudocolors using a “ratio” lookup table. Time course of PIP-SHOW fluorescence was calculated for regions of interest (ROI) inside the imaged cell. The anti-CD3/CD28- coated beads used for T cell stimulation are fluorescent in both channels; however, that fluorescence does not change. The F500/F420 ratio of the beads was set as 1 and all the ratio stacks were normalized to this value. For PI3K activity monitoring, different quantification strategies were applied for the NIH-3T3 cells and for the lymphocytes. For NIH-3T3 cells, two ROI were set up for each cell corresponding to the cytoplasm and to the PM. Distribution of the probe was calculated as Value=(Fmem − Fcyt)/Fcyt. Fmem is a mean value of the fluorescence intensity in a ROI associated with the plasma membrane. Fcyt is a mean fluorescence value in a ROI associated with the cytoplasm. Application of this equation to either 420 or 500 nm channels gives similar results. Since T_h cells are highly motile, it turns to be very time consuming to draw ROIs representing IS and the cytoplasm manually for each frame. We therefore utilized different strategy based on bright pixels count. During accumulation of the probe at the IS, the percentage of high-intensity pixels increased. Therefore, if the low- and medium-intensity pixels are thresholded and set to “Not-a-Number,” dynamics of the mean value of the remaining bright pixels shows dynamics of PI3K activity in the IS. However, in case of PIP-SHOW the brightness of the probe in either channel depends also on H₂O₂. The influence of H₂O₂ can be removed by dividing each frame in the stack by the mean value (in the same frame) of the ROI drawn in the cytoplasm of the cell. To enable this procedure, we designed a simple ImageJ plugin named “Divide by ROI” (Supplementary Data). Being applied to the 420-nm stack (420 nm channel brightness is less dependent on H₂O₂ compared to 500 nm channel), the plugin divides each frame by the mean value of the ROI in the same frame. After that, 32-bit images were thresholded leaving upper 50% (by intensity) of the pixels visible. Mean intensity profile over time gives PI3K dynamics in relative units.

Footnotes

Acknowledgments

We thank Christian Junker for help with imaging. This work was supported by the Russian Foundation for Basic Research (10-04-01561-a, V.V.B; 11-04-12187-ofi, S.L); the Ministry of Education and Science of the Russian Federation (16.512.11.2139, 16.740.11.0367); the Measures to Attract Leading Scientists to Russian Educational Institutions program (11.G34.31.0017 to S.L); ESF (TraPPs, C.S); DFG: BO 3643/2-1 to I.B., Schu 943/7-1 to C.S.; SFB 894 and GK 845 to M.H.; NI671/3-1, SFB 894 and GK 1326 to B.A.N; SB Cancer to C.S.

Abbreviations Used

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Supplementary Material

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