| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Molecular & Cellular Proteomics 6:1500-1509, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,¶,,**,¶¶
From the Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185 Uppsala, Sweden, ¶ Department of Oncology-Pathology, Cancer Center Karolinska, R8:03, Karolinska Institutet, SE-17176 Stockholm, Sweden, || Olink AB, Dag Hammarskjöldsväg 54 A, SE-75183 Uppsala, Sweden, ** Centre for Image Analysis, Uppsala University, SE-75105 Uppsala, Sweden, and 3rd Medical Department, Hematology-Oncology, Johannes Gutenberg University, 55131 Mainz, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRß. We furthermore visualized tyrosine phosphorylated PDGFRß in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.
PDGFRß is a transmembrane protein-tyrosine kinase that becomes dimerized and autophosphorylated at numerous sites upon binding of the cytokines PDGF-BB (4) or -DD (5), leading to activation of several signaling pathways that promote cell proliferation, motility, and survival (6). Studies of these cellular responses have demonstrated the importance of temporal and spatial control of PDGFR phosphorylation (7, 8). Immunofluorescence (IF) analyses with antibodies directed against phosphorylated PDGFR remains the predominant method to monitor the distribution of PDGFR phosphorylation in situ. Unfortunately this method is limited in sensitivity and selectivity, precluding detailed analysis.
We have recently established a technique referred to as the in situ proximity ligation assay (in situ PLA, previously called P-LISA) and used it to demonstrate in situ protein-protein interactions in cells and tissues (9). In situ PLA is a further development of the proximity ligation strategy (10, 11), a protein detection method combining dual recognition of target proteins by pairs of affinity probes generating an amplifiable DNA reporter molecule that acts as a surrogate marker for the detected protein molecule or interacting molecules. In situ PLA uses rolling circle amplification (RCA) for localized detection of proteins or protein interactions in fixed cells or tissues. Antibodies covalently linked to oligonucleotides are used as proximity probes, forming templates for circularization of two additional oligonucleotides by enzymatic ligation. This ligation requires coincident binding by two affinity reagents and thereby increases the selectivity compared with single recognition assays. One of the oligonucleotides then serves as a primer for the RCA reaction, amplifying the circular DNA molecule 
1000-fold in 1 h using 
29 DNA polymerase (12). The product represents a bundle of single-stranded DNA composed of tandem repeats of complements of the DNA circle. Individual bundles are easily visualized by hybridization of complementary fluorescence-labeled oligonucleotides. Because in situ PLA gives rise to a signal at the location where the primary antibodies have bound, it is possible to use it to study the location of proteins and protein complexes in tissues and subcellular compartments.
In the present study we generalized the in situ PLA method by using secondary antibodies with attached DNA strands as proximity probes. This approach preserves the sensitivity and selectivity of the in situ PLA method while permitting the use of general, species-specific antibodies as proximity probes with any suitable primary antibody pair. We utilized the in situ PLA for detection of tyrosine phosphorylation of transfected and endogenous PDGFRß in fixed cultured cells. The phosphorylated receptors were clearly visible as discrete signals permitting quantitative analyses. We furthermore visualized tyrosine phosphorylated PDGFRß in tissue sections from fresh frozen human scar tissue.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Immortalized human foreskin fibroblasts (BJ hTert) were grown in minimum essential medium (Invitrogen) containing 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 50 µg/ml gentamicin. Porcine aortic endothelial (PAE) cells, either untransfected or transfected with the PDGFR or -ß (PAE and PAEß), were grown in F-12 Ham's nutrient medium (Sigma) containing 10% FCS, 1% L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Two days before stimulation 23,000 HEK293 cells/well were seeded on Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY) coated with growth factor-reduced Matrigel (BD Biosciences). For BJ hTert cells 15,000 cells/well were seeded. After 24 h the cells were starved for 8 h or overnight in medium containing 0.5% FCS. Subsequently the cells were stimulated with 1–100 ng/ml human PDGF-BB (Peprotech, Rocky Hill, NJ; powder dissolved and stored in 20 mM Hepes, pH 7.4, 0.5 M NaCl, 5 mg/ml BSA (New England Biolabs, Beverly, MA)) for 1 h on ice. Cells on glass slides were washed once in ice-cold PBS and fixed for 1 h in ice-cold 70% ethanol.
5,000 PAE or PAEß cells/well were seeded and 24 h later starved overnight in medium containing 1% FCS following stimulation with or without 100 ng/ml PDGF-BB (Chiron Corporation, Emeryville, CA; powder dissolved and stored in sodium acetate buffer, pH 4.5) on ice for 1 h. Additionally untransfected PAE cells were, after starvation overnight in 1% FCS, incubated with or without 100 µM pervanadate on ice for 1 h. Subsequently the cells were washed once in ice-cold PBS and fixed for 1 h in ice-cold 70% ethanol.
Preparation of Cell Lysates—
106 BJ hTert cells were seeded on a 10-cm dish and after 2 days starved overnight in medium containing 0.5% FCS following stimulation with or without PDGF-BB (1–100 ng/ml) on ice for 1 h. Cells were then lysed in ice-cold lysis buffer (0.5% Triton X-100, 0.5% deoxycholic acid, 150 mM NaCl, 20 mM Tris, 10 mM EDTA, 30 mM sodium pyrophosphate, pH 7.5) supplemented with 200 µM vanadate, 1% aprotinin, and 2 mM PMSF.
SDS-PAGE/Immunoblotting—
Proteins were separated on an 8% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA) by semidry transfer. The membrane was blocked in TBS containing 5% BSA and 0.05% Tween 20 before overnight incubation with anti-PDGFRß antibody (0.12 µg/ml; Cell Signaling Technology, Danvers, MA). Bound antibodies were visualized using ECL (GE Healthcare) after incubation with secondary antibodies conjugated with horseradish peroxidase, and signals were captured with a charge-coupled device camera (FUJIFILM Manufacturing U.S.A., Inc., Greenwood, SC). The membranes were then stripped and reprobed with anti-Tyr(P)-751 PDGFRß antibody (0.8 µg/ml; Cell Signaling Technology) as described above.
Proximity Probe Design—
The proximity probes consist of affinity-purified polyclonal antibodies with oligonucleotides covalently linked via their 5'-ends to each antibody. The non-priming proximity probes were composed of donkey anti-mouse antibody (catalog number 715-005-150, Jackson ImmunoResearch Laboratories, West Grove, PA) with the covalently linked amine-modified oligonucleotide (NH2-AAA AAA AAA AGA CGC TAA TAG TTA AGA CGC TT[U UU] (the sequence within the brackets is 2'O-methyl-RNA); Trilink BioTechnologies, San Diego, CA), and for the RCA priming proximity probes donkey anti-rabbit antibodies (catalog number 711-005-152, Jackson ImmunoResearch Laboratories) were covalently linked to the amine-modified oligonucleotide (NH2-AAA AAA AAA ATA TGA CAG AAC TAG ACA CTC TT; Trilink BioTechnologies). The proximity probes were conjugated by Solulink, San Diego, CA, using hydrazone linkage.
In Situ PLA Analysis of PDGFRß in Cultured Cells—
Glass slides with ethanol-fixed cells were blocked in 20% goat serum (Invitrogen), 2.5 ng/µl sonicated salmon sperm DNA (GE Healthcare), 2.5 mM L-cysteine (Sigma), 50 µg/ml RNase A (Promega, Madison, WI), 0.1% Tween 20, 5 mM EDTA in PBS for 2 h at 37 °C. Afterward slides were rinsed once with PBS, 0.1% Tween 20 before primary antibodies mouse anti-phospho-PDGFRß (Tyr(P)-751) (0.16 ng/µl; catalog number 3166, Cell Signaling Technology) and rabbit anti-PDGFRß (2 ng/µl; catalog number 3169, Cell Signaling Technology) in 20% goat serum, 2.5 ng/µl sonicated salmon sperm DNA, 2.5 mM L-cysteine, 0.1% Tween 20 in PBS containing 5 mM EDTA were applied and incubated overnight at 4 °C.
Unbound primary antibodies were removed by washing the slides three times for 2 min each in PBS with 0.1% Tween 20. After the washing steps the proximity probes were prepared by separately incubating 0.75 ng/µl non-priming proximity probe and 0.15 ng/µl RCA priming proximity probe (final concentrations) per reaction in PBS, 5 mM EDTA, 20% goat serum, 2.5 ng/µl sonicated salmon sperm DNA, 2.5 mM L-cysteine, 1.5 mM sulfobenzoaldehyde (Solulink), 0.05% Tween 20 for 30 min at room temperature. After incubation the proximity probes were mixed, applied to the glass slide, and incubated for 1 h at 37 °C. The slides were washed in 10 mM Tris-HCl, pH 7.5, 0.1% Tween 20 for 5 min, twice in TBS, 0.05% Tween 20 for 2 min, and rinsed once with ligation buffer (10 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate). All subsequent washing steps were done in TBS, 0.05% Tween 20 for 2 min if not stated otherwise. Hybridization with 125 nM circularization oligonucleotide probes (5'phosphate-CTA TTA GCG TCC AGT GAA TGC GAG TCC GTC TAA GAG AGT AGT ACA GCA GCC GTC AAG AGT GTC TA and 5'-phosphate-GTT CTG TCA TAT TTA AGC GTC TTA A (Eurogentec, Seraing, Belgium)) in ligation buffer with 250 mM NaCl, 0.25 µg/µl BSA, and 0.05% Tween 20 was done for 30 min at 37 °C. After three washes the slides were rinsed once with ligation buffer. The circularization probes were ligated in ligation buffer containing 250 mM NaCl, 0.05 unit/µl T4 DNA ligase (Fermentas, Vilnius, Lithuania), 1 mM ATP (Fermentas), 0.25 µg/µl BSA, and 0.05% Tween 20 for 30 min at 37 °C. Prior to RCA the slides were washed three times and rinsed once in 29 polymerase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, pH 7.5). RCA was allowed to proceed for 90 min at 37 °C in 29 polymerase buffer containing 0.125 unit/µl 29 DNA polymerase (Fermentas), 1 mM dNTPs (Fermentas), 5% glycerol (USB Corp., Cleveland, OH), 0.25 µg/µl BSA, and 0.05% Tween 20. After three additional washing steps 6.25 nM fluorescence-labeled probe Alexa 555-CAG TGA ATG CGA GTC CGT CT (MWG-BIOTECH, Ebersberg, Germany) was hybridized to the single-stranded RCA product in 2x SSC, 0.25 µg/µl BSA, 7.5 ng/µl poly(A) (Sigma), 0.05% Tween 20 for 30 min at 37 °C. The slides were then stained with a mouse anti-actin antibody (Cederlane, Hornby, Ontario, Canada), subsequently incubated with FITC-labeled rabbit anti-mouse antibody (Jackson ImmunoResearch Laboratories), and in the end counterstained with Hoechst 33342 (Sigma).
In Situ PLA Analysis of PDGFRß in Fresh Frozen Tissue—
Fully anonymized human tissue samples were obtained from the Fresh Tissue Biobank at the Department of Pathology, Uppsala University Hospital, in accordance with the Swedish Biobank Legislation. Tape-transfer frozen sections (4 µm thick; CryoJane, Instrumedics Inc., Hackensack, NJ) from fresh frozen human scar tissue were fixed in 70% ethanol on ice for 1 h. In situ PLA reactions were done as described above for the cultured cell experiments besides the following modifications of antibody and enzyme concentrations: 0.6 ng/µl mouse anti-phospho-PDGFRß (Tyr(P)-751) antibody and 2 ng/µl rabbit anti-PDGFRß were applied as primary antibodies. Non-priming proximity probe (1.9 ng/µl) and RCA priming proximity probe (0.43 ng/µl) were used. RCA was performed using 1 unit/µl 29 DNA polymerase. Counterstaining with mouse anti-actin antibody was omitted.
Image Analysis—
The number of in situ PLA signals per cell was counted by semiautomated image analysis. Fully automated cell delineation was initiated by identifying cell nuclei using the signal from the nuclear Hoechst staining (blue). After intensity thresholding, touching nuclei were separated using a combination of distance transformation and watershed segmentation (15). The image channel showing actin staining (green) was thereafter filtered to enhance regions of high intensity variance, i.e. the cytoplasms. Each nuclear delineation was thereafter allowed to expand within these regions to detect the edges of its surrounding cytoplasm. A distance threshold limited the expansion in cases of poor actin staining. Cells whose nuclei were cut by the image border were excluded from the analysis. In situ PLA signals were counted by enhancing pointlike signals and defining a true signal as a local intensity maximum above a background threshold (16). The same input parameters were used throughout all experiments. The methods for image analysis were implemented in Matlab (MathWorks Inc., Natick, MA). Fully automated delineation of cytoplasms did not always give a satisfactory result, and some delineations were corrected manually using the interactive tool in Visiopharm Integrator System (Visiopharm, Horsholm, Denmark).
|
RESULTS |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
Enumeration of Phosphorylated PDGFRß in Single Cells—
In unstimulated HEK293 cells stably overexpressing PDGFRß, minimal signs of specific phosphorylation of the PDGFRß were detected in the absence of PDGF-BB stimulation (Fig. 2a). By contrast, in cells treated with PDGF-BB the amount of signals was dramatically increased (Fig. 2b). However, a heterogenous pattern of staining was observed because some cells had lost the expression of the receptor (as confirmed by normal IF, data not shown). The striking difference in the amount of in situ PLA signals between the two populations attests to the high selectivity of the method (Fig. 2, b and g). Moreover when expression of PDGFRß in HEK293 cells was suppressed using ATC in wild-type HEK293 cells or when one of the primary antibodies was omitted, in situ PLA signals were found in negligible amounts regardless of whether the cells were stimulated with PDGF-BB or not (Fig. 2, c–g). A particular advantage of the in situ PLA method is that individual molecules are detected and thus can be enumerated by digital analysis. We therefore recorded the relative number of phosphorylated receptors per cell in the different investigated populations, revealing distinct differences in the amount of signals between the PDGF-BB-stimulated HEK293-PDGFRß cells and the controls (Fig. 2g).
|
and ß, could be distinguished we used PAE cells stably expressing either of the two receptors. Both isoforms are stimulated equally well by PDGF-BB (17). Using in situ PLA we detected PDGFRß phosphorylation in PDGF-BB-stimulated PAE cells expressing the ß-isoform of the receptor (Fig. 3b) but not in the unstimulated cells (Fig. 3a). Similarly only negligible amounts of signals were detected in cells expressing the -isoform of PDGFR regardless of whether the cells had been stimulated with PDGF-BB or not (Fig. 3, c and d). Enumeration of fluorescent objects revealed the same clear differences in the amount of signals between the PDGF-BB-stimulated PAEß cells and the controls, confirming the ability of in situ PLA to clearly distinguish the receptor isoforms (Fig. 3e).
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
We demonstrated herein that in situ PLA is suitable to detect stimulation-dependent phosphorylation of PDGFRß in individual cells with high selectivity and sensitivity. The brightly fluorescent RCPs allowed unbiased enumeration of phosphorylated PDGFRß molecules in individual cells using dedicated software, revealing intracellular distribution and intercellular variation in the investigated cell populations. All negative control experiments resulted in negligible amounts of signals, whereas abundant signals were observed in positive experiments. Titration of primary antibodies is required to minimize nonspecific signals due to effects such as adsorption of antibodies to surfaces. Because in situ PLA signals depend upon having two antibodies bound to a protein or a protein complex, non-saturating levels of antibodies will decrease the amount of obtained signals as many of the targets only will be occupied with one antibody. On the other hand a too high concentration may increase the probability of nonspecific adsorption of antibodies thereby increasing the background of false positive signals. In general, we have found that a concentration of antibodies that will produce good results in immunofluorescence also works well for in situ PLA. The method of fixation of cells and tissues as well as the method for antigen retrieval also needs to be determined for compatibility with the primary antibody pair used.
Some of the HEK293 cells transfected with PDGFRß had lost their expression during cultivation, resulting in a mixture of cells positive and negative for receptor expression. The striking difference in the amount of in situ PLA signals between the two populations attests to the high selectivity of the method.
It has proven difficult to distinguish between the phosphorylated isoforms of the receptor by normal IF and immunohistochemistry due to antibody cross-reactivity (19),3 whereas in situ PLA offers greater selectivity due to the requirement for two binding events for detection. Furthermore because in in situ PLA RCA is used to amplify the signal it is straightforward to distinguish between RCPs and any autofluorescence or fluorescence from nonspecifically bound detection probes. Using PAE cells that expressed either the - or the ß-isoform of the PDGF receptor, we confirmed that the detected signals were specific for the ß-isoform with no cross-reactivity for PDGFR. Only cells expressing PDGFRß and stimulated with PDGF-BB yielded any signals even though cells expressing PDGFR were stimulated to the same extent.
Experiments using the BJ hTert cell line served to demonstrate that the method also can detect phosphorylated forms of the endogenous PDGFRß upon stimulation with PDGF-BB. There was good agreement between measurements of signals in response to variable amounts of PDGF-BB as determined by in situ PLA and by immunoblotting. PAEß cells expressed about half the amount of receptor compared with the transfected HEK293 cells, but they revealed only one-tenth as many in situ PLA signals. This might be due to differences in levels of phosphatases between the cell lines that may affect the extent of ligand-induced receptor phosphorylation. Concerning quantification of the signals it is noted that the in situ PLA signal is proportional to the real amount of activated receptors as indicated by the comparison with the results from immunoblotting analyses. However, it is also clear that only a fraction of activated receptors gives a signal. This is most likely reflecting less than 100% efficiency in the multiple steps of the procedure, including binding of antibodies, oligonucleotide ligation, and amplification.
Reliable and sensitive detection of protein-tyrosine kinase receptor phosphorylation by in situ PLA in cultured cells and in tissue sections is a new tool of great potential value in basic research and in histopathology. We have previously demonstrated that the proximity ligation mechanism can be extended to using sets of three antibodies (9, 20). This could provide unique opportunities to investigate constellations of several phosphorylations or other PTMs in individual protein molecules, something that would present great difficulties using methods like mass spectrometry.
The in situ PLA method should be suitable to investigate pathophysiological processes in e.g. inflammatory and neoplastic diseases, and it may be of value in the development of PDGFR inhibitors and for predicting the clinical response to tyrosine kinase inhibitors in patients. Moreover the in situ PLA technique could be used to visualize any functional state of proteins in a cell, provided that suitable affinity probes are available, including other PTMs and interactions with other proteins or other macromolecules. On the strength of its selectivity and sensitivity and its potential to investigate inter- and intracellular differences in cells and fresh frozen tissues, in situ PLA thus offers unique possibilities in studies of protein functions in basic and clinical research.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Published, MCP Papers in Press, June 12, 2007, DOI 10.1074/mcp.M700166-MCP200
1 The abbreviations used are: PTM, post-translational modification; ATC, anhydrotetracycline hydrochloride; BJ hTert, immortalized human foreskin fibroblasts; HEK, human embryonic kidney; HA, human influenza hemagglutinin; IF, immunofluorescence; PAE, porcine aortic endothelial; PAE, PAE cells expressing PDGFR; PAEß, PAE cells expressing PDGFRß; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PLA, proximity ligation assay; RCA, rolling circle amplification; RCP, rolling circle product. 
2 K. Pardali, unpublished data.
3 J. Paulsson, unpublished data.
* This study was supported in part by grants from the EU-FP6 (MolTools and Enlight) and from the Swedish Research Councils for Medicine and for Natural Sciences and Technology. The Fresh Tissue Biobank at the Department of Pathology, Uppsala University Hospital, is supported by the National Biobank Platform funded by Wallenberg Consortium North and Swegene. U. Landegren and M. Gullberg are cofounders of the company Olink AB, which exploits the PLA technology under the name DuoLinkTM and holds the right to patents covering PLA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work and are listed in alphabetical order.
Supported by grants from the Swedish Cancer Society, Swedish Research Council, Cancerföreningen, and Gustav Vs Jubileumsfond.
¶¶ To whom correspondence should be addressed. Tel.: 46-18-4714868; Fax: 46-18-4714808; E-mail: ola.soderberg{at}genpat.uu.se
|
REFERENCES |
|---|
|
TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. H. Vermeer, R. van Doorn, R. Dijkman, X. Mao, S. Whittaker, P. C. van Voorst Vader, M.-J. P. Gerritsen, M.-L. Geerts, S. Gellrich, O. Soderberg, et al. Novel and Highly Recurrent Chromosomal Alterations in Sezary Syndrome Cancer Res., April 15, 2008; 68(8): 2689 - 2698. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Journal of Biological Chemistry |
| Journal of Lipid Research | ASBMB Today |
