Combination of two techniques pinpointed distinct activities of different drugs on same molecular pathway.
Scientists have developed a dual technique that allows the simultaneous visualization of protein modifications and downstream mRNA production that occur as a result of cell signaling at the single-cell level. The Uppsala University team say that the approach can also highlight variations in levels of protein phosphorylation and gene expression between neighboring cells and pinpoint where in a signaling pathway a particular drug acts.
Ola Söderberg, Ph.D., and colleagues at the Uppsala University’s Rudbeck Laboratory in Sweden, describe their approach in PLoS One. The paper is titled “Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells.”
Studies on protein-protein interactions, protein modifications, and mRNA-expression levels have to date been performed primarily in bulk populations rather than in individual cells due to restrictions in detection sensitivity and specificity of the methods available, Dr. Söderberg’s team reports. Consequently, while these methods yield average information about molecular pathways, they can’t detect intercellular variations in response that occur during cell-cell signaling.
The Swedish researchers had previously developed two techniques to facilitate the investigation and visualization of molecular events at the single-cell level in fixed cells or tissues. In situ proximity ligation assay (in situ PLA) enables the visualization of endogenous protein interactions and post-translational modifications in single cells. Padlock probes allow the detection of DNA and individual mRNA molecules in situ.
In their latest research the team combined the in situ PLA and padlock probe platforms. Their test system for the technology involved monitoring events that occur as a result of stimulating platelet derived growth factor receptor beta (PDGFRβ) using PDGF-BB.
On stimulation by PDGF-BB, PDGFRβ is dimerized and auto-phosphorylated at several sites, setting off a cascade that includes internalization of the receptor, activation of ERK, and upregulation of downstream mRNA targets, which eventually activates the RAS-RAF-MEK-ERK pathway. What they hoped to be able to visualize was phosphorylation of PDGFRβ and increased mRNA levels of the downstream target DUSP6.
The researchers first used the in situ PLA and padlock probe techniques separately to confirm that the systems could efficiently detect PDGFRβ phosphorylation and DUSP6 upregulation, respectively. They then combined the techniques to visualize both types of molecular events in individual fixed cells.
As hoped, the combined methods not only correctly detected the PDGFRβ phosphorylation following PDGF-BB stimulation and the downstream upregulation of DUSP6 mRNA levels but also showed there was considerable variation in response between cells, especially with respect to the PDGFRβ phosphorylation. This cell-to-cell heterogeneity would have gone undetected if an averaging method was used on a population of cells, the authors note.
It was evident that signal levels dropped about 50% when the two techniques were combined, but this was most likely because the combined protocol “is longer and involves more washing steps than individual detection,” the team writes.
The next stage in testing the approach was to treat cells with drugs that target different points in the PDGFRβ stimulation response pathway to see if the molecular effects could be pinpointed. To this end the researchers separately examined the effects on PDGFRβ stimulation of Gleevec, a tyroskine kinase inhibitor that targets PDGFRβ directly, and 5-iodotubercidin, which inhibits ERK2 activation downstream of PDGFRβ but upstream of DUSP6 expression.
As expected, the dual visualization approach correctly showed that Gleevec eliminated the increase in signals for all molecular events relating to PDGF-BB stimulation, including PDGFRβ phosphorylation and the expression of downstream genes. After treating cells with 5-iodotubercidin, on the other hand, signals relating to DUSP6 upregulation were eliminated due to the drug’s effect on ERK2 activation but phosphorylation of PDGFRβ was unaffected.
“The combined method for detection of individual protein modifications and individual mRNA molecules in single cells in situ presented here may be a valuable tool to enable broader studies of signaling pathways,” the authors state. “The main advantage with the strategy described herein lies in simultaneous targeting of multiple nodes in a signaling cascade to determine how a drug effects the propagation of the signals.”
Moreover, they suggest, because the method is based on imaging, differences between individual cells with respect to target molecule location, amount, and modifications can also be investigated along with cell morphology.