Anchoring of FRET Sensors—A Requirement for Spatiotemporal Resolution

Abstract

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

Rhynchosia volubilis Promotes Cell Survival via cAMP-PKA/ERK-CREB Pathway

“…In particular, they have the advantage of providing accurate information through real-time spatiotemporal data in living cells, which is highly beneficial for verifying the efficacy and activity of drugs. Access to a wide range of fluorescent materials, in conjunction with improved, easy-to-use, and yet very sophisticated microscopes and spectrometers, has made FRET a prominent technique for biosensing [70]. The unique ability of FRET to probe nanoscale inter-and intramolecular separation distances has also led to a rapidly growing field of structural FRET studies of biomolecules and biological complexes [71].…”

Deblurring Signal Network Dynamics

“…52 Anchoring of sensors to stable scaffolds can reduce this limitation. 52 In addition, computational modeling can help to integrate information from distinct measurements into a unified model that also can account for experimental limitations. 7,42 Typical sensors that are used to measure protein activity states detect changes in protein conformation or protein interactions using fluorescent resonance energy transfer (FRET) 53,54 or circular-permuted fluorescent proteins (cpFPs).…”

“…It is thus central to use proper controls to critically evaluate what those proxies actually measure and how they might positively or negatively affect the signal network under study. For example, measurements based on substrate-based sensors only convey a blurred picture of the localization of the corresponding enzymatic activity, if sensor diffusion dominates over sensor reaction kinetics . Anchoring of sensors to stable scaffolds can reduce this limitation .…”

“…For example, measurements based on substrate-based sensors only convey a blurred picture of the localization of the corresponding enzymatic activity, if sensor diffusion dominates over sensor reaction kinetics . Anchoring of sensors to stable scaffolds can reduce this limitation . In addition, computational modeling can help to integrate information from distinct measurements into a unified model that also can account for experimental limitations. , …”