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Detecting Activity at Different Length Scales: From Subdiffraction to Whole-Animal Activity Tsz-Leung To*,† and Xiaokun Shu‡ †
Broad Institute, Cambridge, Massachusetts 02139, United States Department of Pharmaceutical Chemistry and Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 94158, United States
‡
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by the complex photochromic behaviors of FPs, such as blinking under certain illumination conditions. One notable example of a photochromic FP-based imaging method, termed reconstituted fluorescence-based stochastic optical fluctuation imaging (refSOFI), was developed by Zhang and colleagues.1 As an extension of PCA, refSOFI is based on the reconstitution of a photoswitchable FP induced by a specific PPI. The reconstituted FP can be detected by subsequent fluctuationbased super-resolution imaging. Zhang and colleagues successfully applied refSOFI to investigate STIM1/ORAI1 interaction at the endoplasmic reticulum (ER)−plasma membrane junctions, showing that stimulation of store-operated Ca2+ entry increases the number of interacting puncta rather than the size of existing puncta. These two mechanisms would otherwise be indistinguishable with diffraction-limited microscopy. However, refSOFI is based on PCA and therefore irreversible. To enable the detection of reversible dynamic activities using super-resolution imaging, Zhang and colleagues further introduced a FRET-like method called fluorescence fluctuation induced by contact (FLINC). FLINC leverages the significant increase in fluorescence fluctuation of TagRFP-T when it is in the proximity of Dronpa.2 As such, FLINC is highly sensitive to the intermolecular distance between TagRFP-T and Dronpa. As a demonstration, Zhang and colleagues created a FLINC-based PKA activity reporter to discover new insights into PKA signaling using super-resolution imaging. Super-resolution imaging of PPIs and cell signaling events has led to biological discoveries that are not accessible by conventional fluorescence imaging. Another uncharted area for imaging-based studies is the characterization of biochemical events in whole animals. Fluorescence imaging of cell signaling events inside intact tissues and organisms represents a great technical challenge. Although FRET-based reporters are widely used in cell culture models, their in vivo use is limited for two main reasons. First, the signal of FRET reporters is weak because of a small fluorescence change of the donor and acceptor fluorophores. Second, fluorescence imaging of living animals is challenging because of tissue autofluorescence, cell heterogeneity, and rapid shape and position changes. Genetically encoded fluorogenic reporters that provide a much higher signal-to-noise ratio are greatly needed for live imaging of whole animals. One important application of whole-
luorescence is a powerful and versatile imaging modality because it provides high spatial and temporal resolution, together with additional benefits such as high sensitivity and spectroscopic flexibility. As such, fluorescent indicators are often utilized in imaging studies to determine where molecules are active within cells. In particular, fluorescent protein (FP)based biosensors, which can be easily targeted to different subcellular locations, have been developed and deployed for probing a large variety of biochemical processes, including protein−protein interactions (PPIs), calcium signaling, kinase activity, and protease activation. Detection and characterization of PPIs are of great importance because protein assemblies perform almost every major biological process. With the use of global proteomic methods, the molecular constituents of many protein assemblies have been depicted. The next step of the quest is to shed light on the spatiotemporal regulation of protein assemblies, a challenge that requires live-cell fluorescence imaging with high molecular precision. Förster resonance energy transfer (FRET) has been widely used to characterize PPIs at the molecular length scale. FRET is based on the interaction between a donor’s emission dipole moment and an acceptor’s absorption dipole moment. Because the dipole− dipole interaction decays rapidly over distance, FRET is effective when the donor and acceptor fluorophores are in close molecular proximity (i.e.,
