Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors
ABSTRACT Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events.
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- "The Ca2+ reporter used here, could readily be replaced by any other GFP-based genetically encoded reporter. Such tools exist to probe pH, calcium, ATP, NADH, cAMP, glutamate, reactive oxygen species, several redox potentials, activity of kinases and phosphatases, and many other modalities (Hung et al., 2011; Mehta and Zhang, 2011; Depry et al., 2013; Tantama et al., 2013). "
ABSTRACT: The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.Frontiers in Physiology 09/2014; 5:344. DOI:10.3389/fphys.2014.00344 · 3.50 Impact Factor
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- "Type, density, and timing of labeling can be controlled by tissue-specific expression of the recombinase. Genetically encoded fluorescent protein-based biosensors can be used to study a broad assortment of signaling molecules and networks (Depry et al., 2013). For example, fluorescent proteins can be used to monitor protein-protein interactions in living cells using fluorescent resonance energy transfer (FRET) microscopy (Stepanenko et al., 2011; Day and Davidson, 2012). "
ABSTRACT: Corticospinal motor neurons (CSMN) have a unique ability to receive, integrate, translate, and transmit the cerebral cortex's input toward spinal cord targets and therefore act as a "spokesperson" for the initiation and modulation of voluntary movements that require cortical input. CSMN degeneration has an immense impact on motor neuron circuitry and is one of the underlying causes of numerous neurodegenerative diseases, such as primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), and amyotrophic lateral sclerosis (ALS). In addition, CSMN death results in long-term paralysis in spinal cord injury patients. Detailed cellular analyses are crucial to gain a better understanding of the pathologies underlying CSMN degeneration. However, visualizing and identifying these vulnerable neuron populations in the complex and heterogeneous environment of the cerebral cortex have proved challenging. Here, we will review recent developments and current applications of novel strategies that reveal the cellular and molecular basis of CSMN health and vulnerability. Such studies hold promise for building long-term effective treatment solutions in the near future.Frontiers in Neuroanatomy 03/2014; 8:16. DOI:10.3389/fnana.2014.00016 · 4.18 Impact Factor
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- "Genetically encoded probes have become powerful tools for fluorescent analysis of the function and concentration of multiple intracellular ions and proteins (Bregestovski and Arosio, 2012; Depry et al., 2013; Perron et al., 2012). GFP derivatives with different colors have been successfully used to monitor Ca 2+ (Miyawaki et al., 1997; Ohkura et al., 2012), pH (Kneen et al., 1998; Llopis et al., 1998; Miesenbock et al., 1998; Li and Tsien, 2012) and protein–protein interactions (Heim, 1999). "
ABSTRACT: Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.Frontiers in Molecular Neuroscience 05/2013; 6:11. DOI:10.3389/fnmol.2013.00011 · 4.08 Impact Factor