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Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

Bottom Line: This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product.In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry.As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

ABSTRACT
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

No MeSH data available.


Related in: MedlinePlus

In situ prox-HCR.(a–d) Technical controls for the E-cadherin/β-catenin interaction. Strong membranous signal could be observed in HT29 cells when both primary antibodies were applied (a), while omitting either one of the primary antibodies (b,c) or both (d) did not yield visible signal. Phosphorylation of platelet-derived growth factor receptor-β (PDGFR-β) could be shown in BjHTert cells following stimulation with 100 ng ml−1 (f), while expression of phosphorylated receptor was low in non-stimulated cells (e). ProxHCR was used to visualize the induction of autophagy following starvation and incubation with CoCl2 in Caco cells (g–j). Although untreated cells showed only low basal activity (g) of BCL2-BNIP3 interaction, a highly increased signal could be observed in treated cells (h). The same holds true for LC3-SQSTM1 interaction (i,j). The MEK–ERK interaction could be induced in A431 cells by stimulation with 10 ng ml−1 EGF for 10 min (l), while the non-stimulated cells only showed low basal signal (k). Under the same conditions phosphorylation of Akt could be observed (n), while no phosphorylation was visible in non-stimulated cells (m). In panel o, the phosphorylation of Syk is shown (p: no primary antibodies). Detection of Her2 protein was possible as well and shown in panel q (r: no primary antibodies). Expression of E-cadherin/β-catenin interaction could be observed even in FFPE skin tissue (s), while no signal was generated when primary antibodies were omitted (t). White scale bars, 20 nm.
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f4: In situ prox-HCR.(a–d) Technical controls for the E-cadherin/β-catenin interaction. Strong membranous signal could be observed in HT29 cells when both primary antibodies were applied (a), while omitting either one of the primary antibodies (b,c) or both (d) did not yield visible signal. Phosphorylation of platelet-derived growth factor receptor-β (PDGFR-β) could be shown in BjHTert cells following stimulation with 100 ng ml−1 (f), while expression of phosphorylated receptor was low in non-stimulated cells (e). ProxHCR was used to visualize the induction of autophagy following starvation and incubation with CoCl2 in Caco cells (g–j). Although untreated cells showed only low basal activity (g) of BCL2-BNIP3 interaction, a highly increased signal could be observed in treated cells (h). The same holds true for LC3-SQSTM1 interaction (i,j). The MEK–ERK interaction could be induced in A431 cells by stimulation with 10 ng ml−1 EGF for 10 min (l), while the non-stimulated cells only showed low basal signal (k). Under the same conditions phosphorylation of Akt could be observed (n), while no phosphorylation was visible in non-stimulated cells (m). In panel o, the phosphorylation of Syk is shown (p: no primary antibodies). Detection of Her2 protein was possible as well and shown in panel q (r: no primary antibodies). Expression of E-cadherin/β-catenin interaction could be observed even in FFPE skin tissue (s), while no signal was generated when primary antibodies were omitted (t). White scale bars, 20 nm.

Mentions: To test the feasibility of proxHCR to record PPIs and PTMs in situ, we established a number of assays against known interactions and PTMs in a multitude of different cell lines. Figure 4 shows the results of these assays. The E-cadherin/β-catenin shows a strong membranous staining in HT29 cells when both primary antibodies are applied (Fig.4a), whereas omitting either one or both of the primary antibodies results in no detectable signal (Fig. 4b–d). We can further show that a variety of PPIs and PTMs can be visualized using proxHCR (Fig. 4e–o). Among them are membrane receptors such as phosphoplatelet-derived growth factor receptor-β (PDGFR-β) (Fig. 4e,f), indicators of autophagy (that is, BCL2/BNIP3 (Fig. 4g,h) and LC3/STQM3 (Fig. 4i,j)) and members of prominent receptor tyrosine kinase pathways (MEK/ERK interaction (Fig. 4k,l) and phosphorylation of Akt (Fig. 4m,n)). Phosphorylation of Syk in HG3 cells is also very nicely shown (Fig. 4o,p). The biological controls of the induced interactions still show low basal activity (Fig. 4e,g,i,k,m), whereas the technical controls (omission of primary antibody) do not show visible signal (Supplementary Fig. 3). Even single protein detection is possible using proxHCR (Fig. 4q,r). Here, Her2 is visualized using two primary antibodies and two proximity probes. Furthermore, we can show the feasibility of proxHCR for formalin-fixed paraffin-embedded (FFPE) skin tissue sections, staining for E-cadherin and β-catenin (Fig. 4s,t). We used the interaction between E-cadherin and β-catenin in DLD1 cells and in fresh-frozen colon tissue as a model system to compare proxHCR with in situ PLA (Fig. 5a–d). The results show the same specific pattern of signal localization for in situ PLA and proxHCR in cultured cells (Fig. 5 a,b) and in fresh-frozen colon tissue (Fig. 5c,d).


Proximity-dependent initiation of hybridization chain reaction.

Koos B, Cane G, Grannas K, Löf L, Arngården L, Heldin J, Clausson CM, Klaesson A, Hirvonen MK, de Oliveira FM, Talibov VO, Pham NT, Auer M, Danielson UH, Haybaeck J, Kamali-Moghaddam M, Söderberg O - Nat Commun (2015)

In situ prox-HCR.(a–d) Technical controls for the E-cadherin/β-catenin interaction. Strong membranous signal could be observed in HT29 cells when both primary antibodies were applied (a), while omitting either one of the primary antibodies (b,c) or both (d) did not yield visible signal. Phosphorylation of platelet-derived growth factor receptor-β (PDGFR-β) could be shown in BjHTert cells following stimulation with 100 ng ml−1 (f), while expression of phosphorylated receptor was low in non-stimulated cells (e). ProxHCR was used to visualize the induction of autophagy following starvation and incubation with CoCl2 in Caco cells (g–j). Although untreated cells showed only low basal activity (g) of BCL2-BNIP3 interaction, a highly increased signal could be observed in treated cells (h). The same holds true for LC3-SQSTM1 interaction (i,j). The MEK–ERK interaction could be induced in A431 cells by stimulation with 10 ng ml−1 EGF for 10 min (l), while the non-stimulated cells only showed low basal signal (k). Under the same conditions phosphorylation of Akt could be observed (n), while no phosphorylation was visible in non-stimulated cells (m). In panel o, the phosphorylation of Syk is shown (p: no primary antibodies). Detection of Her2 protein was possible as well and shown in panel q (r: no primary antibodies). Expression of E-cadherin/β-catenin interaction could be observed even in FFPE skin tissue (s), while no signal was generated when primary antibodies were omitted (t). White scale bars, 20 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490387&req=5

f4: In situ prox-HCR.(a–d) Technical controls for the E-cadherin/β-catenin interaction. Strong membranous signal could be observed in HT29 cells when both primary antibodies were applied (a), while omitting either one of the primary antibodies (b,c) or both (d) did not yield visible signal. Phosphorylation of platelet-derived growth factor receptor-β (PDGFR-β) could be shown in BjHTert cells following stimulation with 100 ng ml−1 (f), while expression of phosphorylated receptor was low in non-stimulated cells (e). ProxHCR was used to visualize the induction of autophagy following starvation and incubation with CoCl2 in Caco cells (g–j). Although untreated cells showed only low basal activity (g) of BCL2-BNIP3 interaction, a highly increased signal could be observed in treated cells (h). The same holds true for LC3-SQSTM1 interaction (i,j). The MEK–ERK interaction could be induced in A431 cells by stimulation with 10 ng ml−1 EGF for 10 min (l), while the non-stimulated cells only showed low basal signal (k). Under the same conditions phosphorylation of Akt could be observed (n), while no phosphorylation was visible in non-stimulated cells (m). In panel o, the phosphorylation of Syk is shown (p: no primary antibodies). Detection of Her2 protein was possible as well and shown in panel q (r: no primary antibodies). Expression of E-cadherin/β-catenin interaction could be observed even in FFPE skin tissue (s), while no signal was generated when primary antibodies were omitted (t). White scale bars, 20 nm.
Mentions: To test the feasibility of proxHCR to record PPIs and PTMs in situ, we established a number of assays against known interactions and PTMs in a multitude of different cell lines. Figure 4 shows the results of these assays. The E-cadherin/β-catenin shows a strong membranous staining in HT29 cells when both primary antibodies are applied (Fig.4a), whereas omitting either one or both of the primary antibodies results in no detectable signal (Fig. 4b–d). We can further show that a variety of PPIs and PTMs can be visualized using proxHCR (Fig. 4e–o). Among them are membrane receptors such as phosphoplatelet-derived growth factor receptor-β (PDGFR-β) (Fig. 4e,f), indicators of autophagy (that is, BCL2/BNIP3 (Fig. 4g,h) and LC3/STQM3 (Fig. 4i,j)) and members of prominent receptor tyrosine kinase pathways (MEK/ERK interaction (Fig. 4k,l) and phosphorylation of Akt (Fig. 4m,n)). Phosphorylation of Syk in HG3 cells is also very nicely shown (Fig. 4o,p). The biological controls of the induced interactions still show low basal activity (Fig. 4e,g,i,k,m), whereas the technical controls (omission of primary antibody) do not show visible signal (Supplementary Fig. 3). Even single protein detection is possible using proxHCR (Fig. 4q,r). Here, Her2 is visualized using two primary antibodies and two proximity probes. Furthermore, we can show the feasibility of proxHCR for formalin-fixed paraffin-embedded (FFPE) skin tissue sections, staining for E-cadherin and β-catenin (Fig. 4s,t). We used the interaction between E-cadherin and β-catenin in DLD1 cells and in fresh-frozen colon tissue as a model system to compare proxHCR with in situ PLA (Fig. 5a–d). The results show the same specific pattern of signal localization for in situ PLA and proxHCR in cultured cells (Fig. 5 a,b) and in fresh-frozen colon tissue (Fig. 5c,d).

Bottom Line: This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product.In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry.As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical center, Husargatan 3, Box 815, SE-75108 Uppsala, Sweden.

ABSTRACT
Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

No MeSH data available.


Related in: MedlinePlus