Single-Cell Kinetics
BioPen in an ideal tool to study the kinetics biochemical processes on a single-cell level giving you full control of the entire experimental procedure using a confined flow that limits exposure to your region of interest.
With its capability to deliver what you want, where and when you want it, you can target individual cells directly in their native environment without affecting any cells in the surrounding. Since there is no required mixing and since you can continuously monitor your sample during exposure, there is no risk of loosing information about fast processes.
Se below for examples of how BioPen has been used to e.g. study receptor or ion-channel activation kinetics in single-cells.
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Single-cell measurements of GPCR activity
G-protein coupled receptors (GPCR) are a class of transmembrane receptor proteins that are involved in a wide range of physiological processes. Central to the classification of the GPCRs is the ability to activate G-proteins following binding of an agonist to receptor. Frizzleds (FZDs) are a ubiquitously expressed type of transmembrane receptor that share many features with GPCRs, but whose inclusion into that category is disputed due to the lack of evidence for functional G-protein activation. In the paper FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs, Hoffmann and co-workers use the BioPen to study single cells that express the receptor FZD5 and show that the protein exhibits the functional hallmarks of a GPCR.
Both the activation of FZD5 and of a G-protein was investigated using fluorescence resonance energy transfer (FRET). BioPen was used to stimulate individual live-cells with the FZD5 agonist WNT-5A. Because BioPen allows rapid exchange of the microenvironment around the cell, the authors were able to record the time-resolved activation of FZD5 in single cells, which occurs 10-20 s after addition of WNT-5A and which appears as a dequenching of the FRET donor. A similar assay was used for G-protein activation, where dissociation of the G-protein subunits was used as a marker. Here, a dequenching of the FRET donor following ligand addition was again observed.
Together, this together shows that WNT-5A not only activates FZD5, but that receptor activation by the agonist also leads to subsequent activation of the G-protein. This completed the link between ligand, receptor and G-protein activation and confirms that FZD5 can be classified as a GPCR.
Single-cell analysis of CXCR4 Receptor signaling
Chemokine receptors are important components in sell signaling, fulfilling a wide range of biological functions. In the paper Kinetic Analysis of the Early Signaling Steps of the Human Chemokine Receptor CXCR4, recently published in the journal Molecular Pharmacology, Prof. Carsten Hoffmann and coworkers at University Hospital Jena use BioPen to investigate the activation of the chemokine receptor CXCR4 by the chemokine ligand CXCL12 on a single-cell level.
The CXCR4/CXCL12 pair is an well-established pharmaceutical target for cancer treatment and the work by Prof. Hoffmann and coworkers add important new knowledge on the complex mechanisms involved in receptor activation and how that affects cell signaling. The detailed insight into receptor activation that their work provide can facilitate the development of new, more specific, drugs against this target.
Ion Channel Kinetics
An adherent CHO cell culture, expressing TRPV1, was used to demonstrate the application of the BioPen (formerly the Multifunctional pipette) for the delivery of ion channel agonist. More specifically, the effects of cholesterol depletion on the pore dilation of TRPV1 were investigated. The ion channels were activated with the agonists; capsaicin or protons (pH 5.5), and the ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies. While using the BioPen to locally perfuse agonists in tandem with YO-PRO, the uptake rate was monitored by measuring the intracellular fluorescence intensity using confocal microscopy. Cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Using localized perfusion enabled data libraries to be created from individual and small clusters of cells.