Dissecting Pathways; in situ Electroporation for the Study of Signal Transduction and Gap Junctional Communication
Electroporation has been used for the introduction of DNA and proteins, as well as various nonpermeant drugs and metabolites into cultured mammalian cells (reviewed in Neumann et al., 2000; Chang et al., 1992). Most electroporation techniques for adherent cells involve the delivery of the electrical pulse while the cells are in suspension. However, the detachment of these cells from their substratum by trypsin or EDTA can cause significant metabolic alterations (Matsumura et al., 1982), while the efficient incorporation of proteins, peptides, or drugs without cellular damage is an especially crucial requirement, since contrary to DNA, no convenient large-scale method exists for the selection of viable from damaged cells or cells where no introduction took place after electroporation, for most proteins or drugs of interest. For these reasons, a number of approaches have been taken to bypass this problem (Kwee et al., 1990; Yang et al., 1995).
This article describes a technique where cells are grown on a glass surface coated with electrically conductive, optically transparent indium-tin oxide (ITO) at the time of pulse delivery. This coating promotes excellent cell adhesion and growth, allows direct visualization of the electroporated cells, and offers the possibility of ready examination due to their extended morphology. The procedures described are applicable to a wide variety of nonpermeant molecules, such as peptides (Giorgetti-Peraldi et al., 1997; Boccaccio et al., 1998; Bardelli et al., 1998), oligonucleotides (Boccaccio et al., 1998; Gambarotta et al., 1996), radioactive nucleotides (Boussiotis et al., 1997), proteins (Nakashima et al., 1999), DNA (Raptis and Firth, 1990), or drugs (Marais et al., 1997). These compounds can be introduced alone or in combination, at the same or different times, in growth-arrested cells or cells at different stages of their division cycle. After introduction of the material, cells can be either extracted and biochemically analysed or their morphology and biochemical properties examined in situ. In a modified version, this assembly can be used for the study of intercellular, junctional communication. The instant introduction of the molecules into essentially 100% of the cells makes this technique especially suitable for kinetic studies of effector activation. Unlike other techniques of cell permeabilization, under the appropriate conditions, in situ electroporation does not affect cell morphology, the length of the G1 phase of serumstimulated cells (Raptis and Firth, 1990), the activity of the extracellular signal regulated kinase (Erk½ or Erk), or two kinases commonly activated by a number of stress-related stimuli, JNK/SAPK and p38hog (Robinson and Cobb, 1997), presumably because the pores reseal rapidly so that the cell interior is restored to its original state (Brownell et al., 1998).
II. MATERIALS AND INSTRUMENTATION
The purity of the material to be electroporated is of paramount importance. Substances such as detergents, preservatives, or antibiotics could kill the cells into which they are electroporated, even if they have no deleterious effects if added to the culture medium of nonelectroporated cells.
Dulbecco's modification of Eagle's medium (DMEM) is from ICN (Cat. No. 10-331-22). Fetal calf serum (Cat. No. 2406000AJ) and phosphate-buffered saline (PBS, Cat. No. 20012-027) are from Gibco Life Technologies. Calf serum is from ICN (Cat. No. 29-131- 54). EGF is from Intergen (Cat. No. 4110-80). HEPES (Cat. No. H-9136), Lucifer yellow CH dilithium salt (Cat. No. L-0259), trypsin (Cat. No. T-0646), MgCl2 (Cat. No. M-8266), Triton (Cat. No. T-6878), deoxycholate (Cat. No. D-5760), EDTA (Cat. No. E-5134), EGTA (Cat. No. E-3889), phenylmethylsulfonyl fluoride (PMSF, P-7626), aprotinin (Cat. No. A-6279), leupeptin (Cat. No. L-2023), benzamidine (Cat. No. B- 6506), dithiothreitol (DTT, Cat. No. D-9779), CaCl2 (Cat. No. C-4901), Tris (Cat. No. T-6791), paraformaldehyde (Cat. No. P-6148), bovine serum albumin (BSA, Cat. No. A-4503), SigmaFast DAB kit (Cat. No. D-9167 and Cat. No. U-5005), and sodium orthovanadate (Cat. No. S-6508) are from Sigma. Extran-300 detergent (Cat. No. B80002), SDS (Cat. No. 44244), NP-40 (Cat. No. 56009), glycerol (Cat. No. B10118), and peroxide (B 80017) are from BDH. CelTakTM is from BD Biosciences (Cat. No. 354240). The cell staining kit, including goat serum, secondary antibody, and avidin-biotin complex, was from Vector Labs (Vectastain kit Cat. No. PK-6101). Rabbit antipeptide antibodies against the double threonine and tyrosine phosphorylated (activated) Erk½ kinase are from Biosource International (Cat. No. 44-680). When stored frozen in aliquots, they are stable for more than 5 years. They are used at 1:500 for immunostaining and 1:10,000 for Western blotting. Three- and 6-cm tissue culture dishes are from Corning or Sarstedt. Please note that only these brands of plates fit the electroporation stand.
The Grb2-SH2 binding peptide is based on the sequence flanking the Y1068 of the EGF receptor (PVPEPmp- INQS, MW 1123). To enhance stability of the phosphate group, the phosphotyrosine analog, phosphono-methylphenylalanine (Pmp), which cannot be cleaved by phosphotyrosine phosphatases yet binds to SH2 domains with high affinity and specificity (Otaka et al., 1994), is incorporated at the position of phosphotyrosine. The Pmp monomer is custom synthesized by Color your enzyme Inc., (Kingston, Ontario, Canada). As control, we used the same peptide containing phenylalanine at the position of Pmp. Peptides are synthesized by the Queen's University Core Facility using standard Fmoc chemistry.
The system for electroporation in situ (Epizap Model EZ-16) can be purchased from Ask Science Products Inc. (Kingston, Ontario, Canada, phone: 613 545-3794). The inverted, phase-contrast and fluorescence microscope, equipped with filters for Lucifer yellow and fluorescein, was from Olympus (Model IX70).
The technique of in situ electroporation can be used equally effectively for large-scale biochemical experiments (Giorgetti-Peraldi et al., 1997; Boccaccio et al., 1998; Bardelli et al., 1998) or for the detection of biochemical or morphological changes in situ (Raptis et al., 2000a). Cells are grown on glass slides coated with conductive and transparent indium-tin oxide (ITO). The cell growth area is defined by a "window" formed with an electrically insulating frame made of Teflon as shown. A stainless-steel electrode is placed on top of the cells resting on the frame and an electrical pulse of the appropriate strength is applied, as illustrated in Fig. 1 (see also chapter 43 by Raptis et al.). The technique can be applied to a large variety of adherent cell types (Brownell et al., 1996). Cells that do not adhere well can be grown and electroporated on the same conductive slides coated with CelTakTM or poly-L-lysine used according to the manufacturer's instructions.
A. Electroporation of Peptides into Large Numbers of Cells for Large-Scale Biochemical Experiments. Use of Fully Conductive Slides
To study the effect of protein interactions in vivo on cellular functions, such complexes can be disrupted through the introduction of peptides corresponding to the proteins' point(s) of contact. An example of this approach is described here.
Growth factors such as the epidermal growth factor (EGF) stimulate cell proliferation by binding to, and activating, membrane receptors with cytoplasmic tyrosine kinase domains. In vitro binding and receptor mutagenesis studies have shown that ligand engagement induces receptor autophosphorylation at distinct tyrosine residues, which constitute docking sites for a number of effector molecules, such as the growth factor receptor-binding protein 2 (Grb2), which are recruited to specific receptors through modules termed Src-homology 2 (SH2) domains (reviewed in Schlessinger, 2000). Grb2 binds to the receptors for PDGF and EGF at a number of sites, an event activating the Sos/Ras/Raf/Erk pathway, which is central to the mitogenic response stimulated by many growth factors. Previous results indicated that a synthetic phosphopeptide corresponding to the Grb2-binding site of the EGF receptor (EGFR, flanking the EGFR tyr1068 PVPE-pY-INQS), when made in tandem with peptides that allow for translocation across the cell membrane, could inhibit EGF-mediated mitogenesis and Erk activation in newt myoblasts induced by 1 ng/ml EGF, but was less effective at 10ng/ml (Williams et al., 1997). To better determine the functional consequences of disrupting the association of Grb2 per se with different receptors in vivo in mammalian cells, we delivered large quantities of this peptide into intact, living NIH3T3 fibroblasts by in situ electroporation.
Spent medium: Grow cells to confluence in DMEM with 10% calf serum. Seven days postconfluence, collect the culture supernatant and dilute 1:1 with fresh DMEM. Growth-arrest cells by incubating in spent medium prepared from the same line.
Lucifer yellow solution, 5mg/ml: To make 10ml, dissolve 50mg Lucifer yellow in 10ml calcium-free DMEM. Stable at 4°C for at least a month.
Peptides: The peptide concentration required varies with the strength of the signal to be inhibited. For the inhibition of the EGF-mediated Erk activation, prepare a solution of 5-10mg/ml (~5-10mM) of the Grb2-SH2-blocking peptide (PVPE-pmp-INQS, MW 1123 Da) in calcium-free DMEM (see Comment 1).
Epidermal growth factor: To make a 10,000× stock solution, dissolve 100 µg of lyophilised EGF in 100 µl sterile water and freeze in 5µl aliquots. Just prior to the experiment, add 1µl stock solution to 10ml calcium-free DMEM (final concentration, 100ng/ ml). The stock solution is stable at -20° or -70°C for up to 2 months.
Lysis buffer: 50mM HEPES, pH 7.4, 150mM NaCl, 10mM EDTA, 10mM Na4P2O7, 100mm NaF, 2mM vanadate, 0.5 mM PMSF, 10µg/ml aprotinin, 10µg/ml leupeptin, 1% Triton X-100.
As shown in Fig. 2A, electroporation of the Grb2- SH2 blocking peptide caused a dramatic reduction in EGF-mediated Erk activation in mouse NIH3T3 cells at growth factor concentrations permitting full receptor stimulation (compare lanes 2 and 3 with lane 4). In addition, electroporation of this peptide reduced EGFmediated [3H]thymidine uptake (Fig. 2B). In contrast, the same peptide had only limited or no effect on Erk activation triggered by HGF, although it could inhibit PDGF signalling (Raptis et al., 2000a). These findings demonstrate that the in situ electroporation approach described can very effectively inhibit growth factorstimulated mitogenesis and thereby detect the differential specificity in the coupling of activated receptor tyrosine kinases to the Erk cascade.
B. Electroporation for the Study of Morphological Effects or Biochemical Changes in situ: Use of Partly Conductive Slides
Assessment of Erk activity by Western blotting following electroporation of the Grb2-SH2 blocking peptide can reveal the involvement of this domain in growth factor-mediated Erk activation. However, to ensure that the treatment itself does not cause cell stress, examination of cellular morphology, in conjunction with measurement of gene product activity by immunocytochemistry, offers a distinct advantage. This approach can demonstrate the specificity of action of the Grb2-SH2 binding peptide, as well as examine the distribution of signal inhibition across the cell layer. An added advantage is that it requires a small number of cells, hence a substantially smaller volume of the peptides (~14µl in the setup shown in Fig. 1A), compared to Western blotting (~140µl for a cell growth area of 32 × 10mm), which could be a significant consideration given their production costs. To precisely assess small background changes in morphology or gene expression levels, the presence of non-electroporated cells side by side with electroporated ones can offer a valuable control and this can be achieved by growing the cells on a conductive slide where part of the coating has been stripped by etching with acids, thus exposing the non-conductive glass underneath. However, as shown in Fig. 3, area a vs b, the slight tinge of the glass combined with the more effective staining of cells growing on ITO (possibly due to a chemical attraction of different reagents to the coating) can create problems in the interpretation of results. In addition, it was found that a number of cell lines grow slightly better on the conductive, ITO-coated glass than the nonconductive area, possibly due to the fact that the ITO-coated surface is not as smooth as glass, thus providing a better anchorage for the growth of adherent cells (Folkman and Moscona, 1978). As a result, cell density may be higher on the conductive than the etched side, which could have important implications if cell growth effects are being studied. It follows that, to assess the effect of the peptide, it is important to compare the staining and morphology of electroporated cells with non-electroporated ones while both are growing on the same type of surface. This was achieved by plating the cells on a slide where the conductive coating was removed in the pattern shown in Fig. 1A (Firth et al., 1997). A thin line of plain glass separates the electroporated and control areas while etching extends to area Fig. 1A, d so that there is no electrical contact between the positive contact bar and area Fig. 1A,c. Application of the pulse results in electroporation of the cells growing in area Fig. 1A, a exclusively, while cells growing in area Fig. 1A,b or c do not receive any pulse. In this configuration, electroporated cells are being compared to nonelectroporated ones, while both are growing on ITO-coated glass. Because the coating is only ~1,600Å thick, this transition zone does not alter the growth of cells across it and is clearly visible microscopically, even under a cell monolayer (Figs. 3 and 4).
Peptide solution: 5-10mg/ml in calcium-free DMEM. See Section III,A and Comment 1
Lucifer yellow solution: 5 mg/ml in calcium-free DMEM. See Section III,A
Epidermal growth factor: 100ng/ml in calcium-free DMEM. See Section III,A.
As shown in Fig. 3, electroporation of the Grb2-SH2 blocking peptide totally inhibited EGF-induced Erk activation (panel A, area "a"), while the control, phenylalanine-containing peptide had no effect (panel C, area "a"). This inhibition was uniform across the cell layer, in agreement with previous results indicating that in situ electroporation can introduce the material into essentially 100% of the treated cells. It is especially noteworthy that this inhibition extends into three to four rows of the adjacent, nonelectroporated cells growing on the nonconductive part of the slide (panel A, area "b", squiggly bracket), probably due to movement of the 1123Da peptide through gap junctions (Raptis et al., 1994). This finding constitutes compelling evidence that the observed inhibition must be due to the peptide rather than an artifact of electroporation. At the same time, as shown by phase-contrast microscopy (panel B), there was no alteration in the morphology of the electroporated cells under these conditions, suggesting that the observed effect is a result of a specific inhibition rather than toxic action. EGF stimulation for up to 30 min after peptide electroporation did not result in lower levels of Erk signal inhibition, indicating that the binding of the peptide to Grb2 is stable during this period of time. As expected, the phenylalanine-containing, control peptide (panels C and D) had no effect on Erk activation. In contrast, the Grb2-SH2 binding peptide had little effect in inhibiting Erk activity triggered by the hepatocyte growth factor (HGF) in NIH3T3 cells expressing the HGF receptor through transfection or in human A549 cells that naturally express this receptor (Raptis et al., 2000a).
The introduction of peptides to interrupt signaling pathways using the modification of in situ electroporation described is a powerful approach for the in vivo assessment of the relevance of in vitro interactions. Results presented in Fig. 2 and 3 clearly demonstrate that an essentially complete and specific inhibition of EGF-dependent Erk activation can be achieved through peptide electroporation. The stepwise dissection of signaling cascades is essential for the understanding of normal proliferative pathways, which could lead to the development of drugs for the rational treatment of neoplasia.
C. Electroporation on a Partly Conductive Slide for the Assessment of Gap Junctional, Intercellular Communication
One of the targets of a variety of signals stemming from growth factors or oncogenes may be membrane channels, which serve as conduits for the passage of small molecules between the interiors of cells. Oncogene expression and neoplasia invariably result in a decrease in gap junctional, intercellular communication (GJIC) (Goodenough et al., 1996). The investigation of junctional permeability is often conducted through microinjection of a fluorescent dye such as Lucifer yellow, followed by observation of its migration into neighboring cells. This is a time-consuming approach, requiring expensive equipment, while the mechanical manipulation of the cells may disturb cellto- cell contact areas, interrupt gap junctions, and cause artefactual uncoupling. These problems can be overcome using a setup where cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of Lucifer yellow, causing its penetration into cells growing on the conductive part of the slide, and migration of the dye to non-electroporated cells growing on the nonconductive area is observed microscopically under fluorescence illumination.
The technique can be applied to a large variety of adherent cell types, including primary human lung carcinoma cells (Tomai et al., 1998; Raptis et al., 1994; Brownell et al., 1996; Vultur et al., 2003).
Lucifer yellow solution: 5 mg/ml in calcium-free DMEM or other growth medium. See Section III.A
Calcium-free growth medium with or without 5% dialysed serum
The concentration of peptide required varies with the strength of the signal to be inhibited. For example, for the inhibition of the HGF-mediated Stat3 activation in MDCK cells, a concentration of 1 µg/ml of a peptide blocking the SH2 domain of Stat3 (PYVNV) is sufficient (Boccaccio et al., 1998), whereas for the inhibition of the EGF-mediated Erk activation in a variety of fibroblasts or epithelial cells, a concentration of 5-10mg/ml (~5-10mM) of the Grb2-SH2 blocking peptide is necessary (Raptis et al., 2000a). The purity of the material is of utmost importance. Peptides must be HPLCpurified because impurities can cause cell death or give unexpected results. The pH of the peptide solution must be neutral, as indicated by the color of the DMEM medium where the peptide is dissolved. If it is too acidic, then it must be carefully neutralised with NaOH. In this case, the salt concentration of the nopeptide controls (DMEM without calcium) must be adjusted to the same level with NaCl because a change in conductivity may affect the optimal voltage required (see Comment 3).
Any peptide which is soluble in DMEM or other aqueous buffer can be very effectively electroporated. Good solubility is especially important because the concentration needed for effective signal inhibition can be as high as 10 mg/ml. It was nevertheless found that, at least for certain applications, the inclusion of DMSO in the electroporation solution at a concentration of up to 5%, which might aid peptide solubility, did not affect results significantly. However, a number of peptides, e.g., peptides made as fusions with the homeobox domain or other membrane translocation sequences, are usually not sufficiently soluble for this application.
As described in detail in Chapter 43 by Raptis et al., to obtain a uniform electrical field intensity over the entire area below the negative electrode, despite the fact that the conductive coating exhibits a significant amount of electrical resistance, the bottom surface of the negative electrode must be inclined relative to the glass surface in a manner proportional to the resistance of the coating. For electroporation of peptides, to minimise the volume of custom-made peptide used, slides with a conductivity of 2Ω/sq, the most conductive commercial grade available, are used. The slides and electrodes come in different sizes, with the biggest cell growth area in this configuration being 32 × 10mm. Depending on the experiment, if larger numbers of cells are required, extracts from two to three slides may be pooled. In this case, the peptide solution can be aspirated and used again. Alternatively, an electrode configuration with two positive contact bars can be employed, as described in Chapter 43 by Raptis et al.
The slides come with the apparatus, individually wrapped and sterile. However, they can be reused many times after washing with Extran-300 detergent while scrubbing with a toothbrush. In this case they must be sterilized with 80% ethanol for 20min and the ethanol rinsed with sterile distilled water prior to plating the cells. Alternatively, the slides can be gassterilized. Do not autoclave. The glass can withstand high temperatures, but autoclaving would damage the Teflon frames.
3. Determination of the Optimal Voltage and Capacitance
Electrical field strength has been shown to be a critical parameter for cell permeation, as well as viability (Chang et al., 1992). It is generally easier to select a discrete capacitance value for a given electroporated area and space between the conductive coating and the negative electrode and then precisely control the voltage. Both parameters depend upon the size of the electroporated area; larger conductive growth areas necessitate higher voltages and/or higher capacitances for optimal permeation. For the 32 × 10 mm cell growth area, some damage to the coating may be noted at the higher voltages necessary if a single pulse is employed. However, using higher capacitance values and multiple pulses with lower voltage settings can yield efficient cell permeation with no damage to the coating, and this treatment is also better tolerated by the cells. For greater growth areas, the dual positive contact bar design described in Chapter 43 by Raptis et al. can be employed.
The optimal pulse strength depends on the strain and metabolic state of the cells, as well as on the degree of cell contact with the conductive surface. Densely growing, transformed cells or cells in a clump require higher voltages for optimum permeation than sparse, subconfluent cells, possibly due to the larger amounts of current passing through an extended cell. Similarly, cells that have been detached from their growth surface by vigorous pipetting prior to electroporation require substantially higher voltages. It is especially striking that cells in mitosis remain intact under conditions where most cells in other phases of the cycle are permeated (Raptis and Firth, 1990). In addition, cells growing and electroporated on CelTakTM-coated slides require substantially higher voltages than cells growing directly on the slide.
The margins of voltage tolerance depend on the size and electrical charge of the molecules to be introduced (Fig. 5). For the introduction of small, uncharged molecules such as Lucifer yellow or peptides, a wider range of field strengths permits effective permeation with minimal damage to the cells than the introduction of antibodies or DNA (Raptis and Firth, 1990; Brownell et al., 1997). For a number of experiments involving cell growth, it may be necessary to electroporate serum-arrested cells. Voltages required are lower, and especially the margins of voltage tolerance were found to be substantially narrower for serumstarved cells compared to their counterparts growing in 10% calf serum (Brownell et al., 1997). Also, it is important to keep all solutions at 37°C, which facilitates pore closure and efficient electroporation. If the material is applied in a medium with a lower salt concentration than DMEM, then the voltages required are lower, presumably due to the hypotonic shock to the cells and to the longer duration of the pulse because of the lower conductivity of the medium. Conversely, electroporation in a hypertonic solution requires higher voltages for optimum permeation.
Cell damage is manifested microscopically by the appearance of dark nuclei under phase-contrast illumination. For most lines this is most pronounced 5-10min after the pulse. Such cells do not retain Lucifer yellow and fluoresce very weakly, if at all (Fig. 6). Despite the fact that every effort is made to make the electric field uniform over the whole cell growth area, the current flow along the border with the etched side is greater than the rest of the conductive surface. For this reason, as the voltage is progressively increasing, damaged cells will appear on this edge first (Fig. 6). Another area receiving a slightly higher current is corners of the window. This slight irregularity has to be taken into account when determining the optimal voltage.
4. Example of the Determination of Optimal Voltage for the Introduction of Peptides
Prepare a series of slides with cells plated uniformly in a 4 × 7mm window on a partly conductive slide (Figs. 1A or 1B). Set the apparatus at 0.5 µF capacitance. Prepare a solution of 5mg/ml Lucifer yellow and electroporate at different voltages (0.5 µF, three pulses) to determine the upper limits where a small fraction of the cells at the border with the etched side (probably the more extended ones) are killed by the pulse, as determined by visual examination under phase-contrast and fluorescence illumination (Fig. 6). Depending on the cells, this voltage can vary from 20 to 40 V. Repeat the electroporation using the peptide solution at different voltages starting at 10 V below the upper limit and at 2 V increments. The Lucifer yellow dye offers an easy way to test for cell permeation.
Results of a typical experiment of electroporation of cells growing on a 32 × 10mm surface are shown in Fig. 5. The Application of three exponentially decaying pulses of an initial strength of 25V from a 32µF capacitor to rat F111 cells growing on a conductive growth area of 32 × 10 mm, resulted in essentially 100% of the cells containing the introduced Lucifer yellow, whereas introduction of a nine amino acid peptide required 30 V and the stable expression of DNA 45 V, respectively, for maximum signal. If a 20µF capacitor is used, the corresponding voltages are ~80-180. Electroporation on 7 × 15 mm slides requires a 2µF capacitor and voltages of 25-50. For etched slides with a conductive area of 4 × 4mm, if a 0.5 µF capacitor and three pulses are used, the voltages are 20-40. However, if a 0.1µF capacitor and six pulses are used, the voltages will be ~30-50.
Under the appropriate conditions, electroporation was shown not to affect the activity of Erk½ or the stress-activated kinases JNK/SAPK and p38hog This was shown by probing with antibodies specific for the activated forms of these kinases (Fig. 7); no activation of JNK/SAPK or p38hog was found under conditions of up to 70V (Figs. 7C and 7E). These kinases were, however, slightly activated at voltages higher than 85 V, when more than 60% of the cells were killed by the pulse (not shown).
The financial assistance of the Canadian Institutes of Health Research, the Canadian Breast Cancer Research Initiative, the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Cancer Research Society Inc. is gratefully acknowledged. AV is the recipient of NSERC and Ontario Graduate studentships, a Queen's University graduate award, and a Queen's University travel grant. HB was the recipient of a studentship from the Medical Research Council of Canada and a Microbix Inc. travel award. We are grateful to Dr. Erik Schaefer of Biosource Int. for numerous suggestions and valuable discussions.
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