ABSTRACT
Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 ß cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in ß cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse ß cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated ß cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.
Subject(s)
Insulin-Secreting Cells/metabolism , Insulin/metabolism , Animals , Biosensing Techniques , Calcium/metabolism , Cell Line , Genes, Reporter , Glucose/pharmacology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hypoglycemic Agents/pharmacology , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Microscopy, Fluorescence , Recombinant Fusion Proteins/metabolism , Tolbutamide/pharmacology , Red Fluorescent ProteinABSTRACT
Optical sensors are powerful tools for live cell research as they permit to follow the location, concentration changes or activities of key cellular players such as lipids, ions and enzymes. Most of the current sensor probes are based on fluorescence which provides great spatial and temporal precision provided that high-end microscopy is used and that the timescale of the event of interest fits the response time of the sensor. Many of the sensors developed in the past 20 years are genetically encoded. There is a diversity of designs leading to simple or sometimes complicated applications for the use in live cells. Genetically encoded sensors began to emerge after the discovery of fluorescent proteins, engineering of their improved optical properties and the manipulation of their structure through application of circular permutation. In this review, we will describe a variety of genetically encoded biosensor concepts, including those for intensiometric and ratiometric sensors based on single fluorescent proteins, Forster resonance energy transfer-based sensors, sensors utilising bioluminescence, sensors using self-labelling SNAP- and CLIP-tags, and finally tetracysteine-based sensors. We focus on the newer developments and discuss the current approaches and techniques for design and application. This will demonstrate the power of using optical sensors in cell biology and will help opening the field to more systematic applications in the future.
