Abstract

Single Cell Analysis of Cellular Communication and Signaling Pathway Activity

Ola Soderberg, Associate Professor/Senior Lecturer, Uppsala University

Measurements in single cells are required to monitor cell signaling, taking in account the differences between individual cells. Further, to understand cellular communication and how the microenvironment influences growth, survival and differentiation, analyses need to be performed in situ keeping relationship between the cells intact. Methods should also be able to simultaneously measure multiple types of biomolecules, such as DNA, RNA and proteins, but modifications and interactions of these. During the last few years several methods have been developed in our group, facilitating such analyses. The activity status of a protein or signaling pathway can be visualized with in situ Proximity Ligation Assays (in situ PLA) using a pair of antibodies targeting an interacting protein pair. These antibodies are equipped with DNA oligonucleotide that template the creation of a circular DNA molecule. This surrogate marker for the interaction can then be amplified by rolling circle amplification (RCA) and be detected, with a single-molecule resolution, in fixed cells or tissues. Analysis of mRNA can be performed using padlock probes, providing single nucleotide resolution. Multiplexed analysis combining in situ PLA and padlock probes is a powerful tool for analysis of signaling networks that will play an important role in future diagnostics.