FRET-based activity biosensors to probe compartmentalized signaling.
TL;DR: Approaches of genetically encoded fluorescent biosensors with a focus on understanding compartmentalized signaling of kinase and second-messenger dynamics and methods for tracking activity dynamics of signaling molecules with high spatiotemporal resolution in the native cellular environment are discussed.
Abstract: The ability of a cell to properly respond to environmental changes is important for cell growth and survival. Appropriate cellular responses are mediated through exquisitely organized regulatory networks, which consist of highly dynamic signaling molecules. For the purpose of achieving signaling specificity and efficiency, it is crucial that activities of these signaling molecules are spatially compartmentalized within a cell. For example, the intracellular second messenger, cyclic AMP (cAMP), is known for its ability to modulate a wide variety of fundamental cellular processes, including metabolic, electrical, cytoskeletal, and transcriptional responses. 3] To account for specific regulation of these diverse processes, subcellular compartmentation of cAMP signaling was suggested more than 20 years ago. As shown in cardiomyocytes, the binding of two extracellular ligands, prostaglandin E1 (PGE1) and isoproterenol, to different G protein-coupled receptors (GPCR) results in similar levels of cellular cAMP accumulation but distinct physiological outcomes. Isoproterenol stimulation enhances contractile activity through activation of particulate or membrane-bound cAMP-dependent protein kinase (PKA), whereas PGE1 stimulation causes no changes in contractile activity, correlated with cAMP elevation and PKA activation in the soluble fraction of heart homogenates. 5] Recent studies have provided new evidence for compartmentalized cAMP signaling at different levels of the signaling cascade. At the level of cAMP, compartmentalized phosphodiesterases (PDE) restrict cAMP accumulation within domains in correspondence with the transverse tubule region to limit the activation to a specific population of PKA. At the PKA level, anchored by A-kinase anchoring proteins (AKAP), different pools of PKA lie in proximity to distinct substrates to modulate their phosphorylation. Therefore, it appears that cAMP accurately mediates various cellular processes through a signaling network in which the action of each signaling molecule is tightly controlled in defined subcellular compartments. cAMP compartmentation is not an exception to kinaseand second messenger-mediated signal transduction. It has become increasingly clear that spatial compartmentalization is a general theme in signal transduction and plays pivotal roles in ensuring specific signal processing by various signaling pathways, such as Ca, phosphoinositide, mitogen-activated protein kinase (MAPK), and Rho GTPase pathways. Given the dynamic nature of signaling molecules as well as the critical involvement of cellular parameters and constraints in forming various signaling compartments, a better understanding of compartmentalized signal transduction requires methods for tracking activity dynamics of signaling molecules with high spatiotemporal resolution in the native cellular environment. To meet this challenge, a series of genetically encoded fluorescence resonance energy transfer (FRET)-based activity biosensors have been developed for tracking dynamics of various signaling molecules, such as GTPases, protein kinases, second messengers, and membrane receptors. Other FRET-based approaches, including visualization of enzyme–substrate interactions by utilizing fluorescent protein-tagged enzyme and fluorophore-labeled substrate have also been applied to study signaling compartmentation. In this short review, we discuss applications of genetically encoded fluorescent biosensors with a focus on understanding compartmentalized signaling of kinase and second-messenger dynamics. For general reviews regarding the development and application of these biosensors, please refer to recent review articles. 17–21]
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TL;DR: The basic principles and applications of FRET in chemistry, biology, and physics are discussed and the recent improvements in optical techniques facilitate the measurements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecular interactions.
Abstract: Forster resonance energy transfer (FRET) in association with the recent advancements in optical techniques provides a way to understand the detailed mechanisms in different biological systems at the molecular level. Improvements in wide-field, confocal and two-photon microscopy facilitate the measurements of two-dimensional spatial distribution in steady-state as well as dynamic bimolecular interactions. In the recent decade, FRET became an exceptional fluorescence-based technique due to its potential advantages for studying the biological processes in living cells and more for spatial resolution at nanometer scale. In particular, FRET investigations have shown that biomolecules adopt different conformational structures to perform their functions. In this review, the basic principles and applications of FRET in chemistry, biology, and physics are discussed. Along with, the recent improvements in fluorophore design and labeling and FRET measurement methods are briefly mentioned.
268 citations
TL;DR: A review of the dynamics of plexcitonics in various composite systems and an overview of the latest theoretical and experimental developments in the field of PLC can be found in this article.
Abstract: The nanoscale confinement and coupling of electromagnetic radiation into plexcitonic modes has drawn immense interest because of the innovative possibilities for their application in light harvesting and light emitting devices (LEDs). Plexcitons arise from the coupling between two types of quasiparticles, plasmons and excitons, and can be distinguished by the strength of the coupling into strong and weak coupling regimes. Plexcitons have been used to modulate the rate of Forster-type resonance energy transfer in quantum dot assemblies and enhance the spontaneous emission rate in quantum dot LEDs. The clearest examples of a plexcitonic enhancement of photocatalytic reaction rates have been evidenced in hybrid systems wherein the strongly bound exciton found in 2D sheet-like semiconductors is coupled to the surface plasmon resonance of close-lying noble metal nanoparticles. Plexcitonic photocatalysts and solar cells aim to increase the lifetime of hot carriers and thereby enhance the quantum yields for energy harvesting. Since plexcitonics requires the placement of plasmonic and excitonic components in close proximity with one another to facilitate their coupling, it provides a rich arena for chemists and materials scientists to form deterministic and non-deterministic arrays and heterojunctions involving noble metal thin films and nanostructures, quantum dots and dye molecules. This review summarizes the dynamics of plexcitons in the various composite systems and provides an overview of the latest theoretical and experimental developments in the field of plexcitonics.
80 citations
TL;DR: A discrete ‘insulating’ function of primary cilia is identified in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and a cellular activity of GPR88 is suggested that might underlie its effects on dopamine-dependent behaviors.
Abstract: A number of G protein-coupled receptors (GPCRs) localize to primary cilia but the functional significance of cilia to GPCR signaling remains incompletely understood. We investigated this question by focusing on the D1 dopamine receptor (D1R) and beta-2 adrenergic receptor (B2AR), closely related catecholamine receptors that signal by stimulating production of the diffusible second messenger cyclic AMP (cAMP) but differ in localization relative to cilia. D1Rs robustly concentrate on cilia of IMCD3 cells, as shown previously in other ciliated cell types, but disrupting cilia did not affect D1R surface expression or ability to mediate a concentration-dependent cAMP response. By developing a FRET-based biosensor suitable for resolving intra- from extra- ciliary cAMP changes, we found that the D1R-mediated cAMP response is not restricted to cilia and extends into the extra-ciliary cytoplasm. Conversely the B2AR, which we show here is effectively excluded from cilia, also generated a cAMP response in both ciliary and extra-ciliary compartments. We identified a distinct signaling effect of primary cilia through investigating GPR88, an orphan GPCR that is co-expressed with the D1R in brain, and which we show here is targeted to cilia similarly to the D1R. In ciliated cells, mutational activation of GPR88 strongly reduced the D1R-mediated cAMP response but did not affect the B2AR-mediated response. In marked contrast, in non-ciliated cells, GPR88 was distributed throughout the plasma membrane and inhibited the B2AR response. These results identify a discrete ‘insulating’ function of primary cilia in conferring selectivity on integrated catecholamine signaling through lateral segregation of receptors, and suggest a cellular activity of GPR88 that might underlie its effects on dopamine-dependent behaviors.
58 citations
Cites background from "FRET-based activity biosensors to p..."
...FRET-based cAMP biosensors have proven very useful for assessing subcellular cAMP dynamics, as reviewed elsewhere [13,14], but we were unable to identify an existing biosensor construct that achieved sufficient ciliary expression to allow reliable detection of cAMP accumulation in this compartment....
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TL;DR: A cell-penetrating peptide biosensor for dynamic monitoring of phosphorylation by Abl kinase based on fluorescence lifetime imaging microscopy (FLIM), which has the potential to circumvent key technological gaps as a new strategy for studying intracellular signaling biology.
Abstract: Many cancers exhibit deregulated activity of protein kinase enzymes, but not all are sensitive to inhibitor drugs, largely because phosphorylation dynamics in complex tissues are not well understood.[1] Live, subcellular analysis can reveal the details of kinase signaling in mixed populations of cells.[2] Current tools to image kinase activity in situ depend on intensity-based measurements (such as fluorescence and Forster resonance energy transfer) that can be limited by spectral bleed-through and photobleaching.[3] We report a cell-penetrating peptide biosensor for dynamic monitoring of phosphorylation by Abl kinase based on fluorescence lifetime imaging microscopy (FLIM).[4] FLIM, which is not confounded by photobleaching or cellular autofluorescence, was applied to detect phosphorylation-dependent fluorophore lifetime shifts (1–2 ns) in intact, living cells (Fig. 1). We established the dependence of the fluorophore lifetime shift on phosphorylation specifically by Abl kinase, mapped the fluorophore intensity and lifetime components to quantify subcellular phosphorylation, and monitored kinase inhibition in real time. This approach should be generalizable to other kinases and provides a new method for interrogating real-time, subcellular signaling activities in cell populations that are not amenable to expression of genetically engineered biosensor proteins.
Figure 1
FLIM to detect phosphorylation-dependent fluorophore lifetime shifts for biosensors in intact, live cells
Measuring subcellular kinase activity in living cells remains a major challenge. Genetically encoded Forster resonance energy transfer (FRET) biosensors can be used for this purpose in simple cell-based assays and basic research applications.[3, 5] These sensors take advantage of binding between phosphorylated sequences and phosphopeptide binding domains to bring two fluorescent proteins close enough for energy transfer to occur. However, expressing genetically engineered proteins in cells has challenges, including a) uniform transfection and expression of protein fluorophores (a roadblock for applications in primary patient-derived cells or tissues) and b) the large labels which can affect substrate function and interaction with a kinase.[5–6] Small molecule fluorophores are able in principle to be less disruptive to function, and many are available for which excitation and emission do not overlap with expressible fluorophores (enabling multiplexed co-localization experiments).[7] These have been applied to detect phosphorylation in cells via fluorescence intensity increases.[8]
Low signal to noise is a limitation of FRET, and intensity-based fluorescence is confounded by photobleaching when experiments are conducted over long time periods, making it difficult to interpret subcellular fluctuations at high spatial and temporal resolution.[6] FLIM is not affected by photobleaching or intensity and has the potential for single molecule monitoring.[4, 9] Also, time-correlated single photon counting FLIM is capable of highly resolved discrimination between species exhibiting very small differences in lifetimes (even sub-nanosecond), facilitating the mapping of exquisite detail in subcellular images. Here we describe the first demonstration of a FLIM-based phosphorylation biosensor technology that has the potential to circumvent key technological gaps as a new strategy for studying intracellular signaling biology.
We combined the delivery of organic fluorophore-tagged kinase substrate peptide probes with time-resolved FLIM to visualize the details of kinase activity in live, intact cells (Fig. 1). The biosensor consists of an Abl substrate peptide containing the “Abltide” substrate sequence[10] tagged with a Cy5 fluorophore and a cell penetrating peptide (Abl-TAT: GGEAIYAAPCCy5GGRKKRRQRRRPQ) (Fig. 2).[11] The substrate portion (bold) is relatively selective for the c-Abl kinase (Abl1) over other tyrosine kinases, however it is phosphorylated by the Abl family member named Abl-related gene (Arg, also known as Abl2).[12] Abl1 and Abl2 are highly homologous and share many functions in normal cells.[13] In this work, “Abl kinase” denotes both Abl1 and Abl2. We used FLIM instrumentation with picosecond pulsing lasers[9a, 9b] to measure Cy5 lifetime for the unphosphorylated biosensor and a phosphorylated derivative in solution and in live cells.
Figure 2
Peptide-based Abl kinase biosensor
In solution, lifetime differences between the phosphorylated and unphosphorylated Abl-TAT peptide species were not significant (see supporting information, Fig. S1a), indicating that phosphorylation alone is not sufficient to elicit a change in the rate of fluorescence signal decay for the Abl-TAT peptide sensor. However, in the presence of c-Abl kinase at 1:1 ratio, robust lifetime differences were observed, and this phenomenon was blocked by pre-incubation of the kinase with higher ratios of unlabelled phosphopeptide (Fig. S1b). This effect likely arises from the more drastic change in the local environment of the fluorophore that could occur upon binding of the phosphopeptide with the protein, probably through the kinase SH2 domain.[14]
Since the physiochemical basis for the lifetime shift was still somewhat unknown, standards were established in NIH3T3 immortalized mouse embryonic fibroblast cells (MEFs)[15] to assess phosphorylation- and Abl kinase-dependence of the lifetime shifts by using three key negative controls (which exhibited lower lifetimes in cells): Cy5 alone, a non-phosphorylatable Y→F peptide sensor analog (Abl-F-mutant) (both in control MEFs expressing Abl kinase) and the Abl-TAT biosensor in Abl(−/−) knockout cells[15] (Fig. 3B, C and E and Fig. S2). Average lifetimes per cell for multiple cells were calculated and plotted to show the distribution of lifetimes observed for the biosensor and each control (Fig. 3G). The distributions were determined to be non-Gaussian, so non-parametric ANOVA with a Dunn’s post-test (described in the Methods section) was used to evaluate statistical significance (P<0.05) for differences in the mean lifetimes. There was no significant difference between the Cy5, Abl(−/−) or Abl-F-mutant experiments, however each of these controls exhibited significantly lower mean lifetimes than both Abl-TAT (in MEFs expressing Abl kinase) and Abl-phospho (positive control). The mean lifetime for Abl-phospho was also significantly longer than that of Abl-TAT, consistent with enrichment of the phosphorylated form of the substrate. These experiments confirmed that the increase in Cy5 lifetime was specific and due to Abl dependent phosphorylation of the biosensor peptide on tyrosine. As another control to support the interpretation of phosphorylation dependence for lifetime increases in cells, we used immunocytochemistry to show colocalization between the Abl-TAT biosensor and phosphotyrosine (see supporting information, Fig. S3).
Figure 3
FLIM mapping of biosensor phosphorylation
We tested sequestration of the biosensor in endosomes by staining for an endosomal marker. Minimal, non-exclusive colocalization of the biosensor with endosomes was observed, indicating that the biosensor peptide was not sequestered (Fig. S3). We also examined peptide degradation, a potential issue in some cell types[16] but not all, as demonstrated from our prior work.[11a] Controls using fluorescence correlation spectroscopy (FCS) measured Cy5 alone vs. the Abl-TAT biosensor in MEFs (supporting information, Figure S4), providing evidence that for MEFs, the signal observed for the Abl-TAT biosensor arose from peptide that was not degraded to free Cy5.[17]
To quantitatively address the distribution and level of biosensor phosphorylation in different subcellular regions we separated the intensity and lifetime components of the signal arising from the FLIM measurements and plotted lifetime values in 2D using MatLab (as shown in Fig. 3A–F). We did not observe phosphorylation-dependent intensity increases for this biosensor either in solution or in cells (Fig. S2A) (in contrast to what has been observed by others).[8a, 18] We then used the Abl-TAT biosensor to image Abl kinase inhibition with the kinase inhibitor imatinib in control MEFs (Fig. 4). MEFs stably expressing a nuclear-enriched Abl kinase mutant, FKBP-Abl(NUK),[15] were also analysed in the presence of imatinib (see time lapse movie shown in Fig. S5). Over the course of 70 min we detected a general trend towards decreased lifetime overall (e.g. Fig. 4 and Fig. S5) and negative lifetime shifts in multiple areas of the cell within the first few minutes of incubation (Fig. S6), consistent with kinase inhibition and dephosphorylation of the biosensor by phosphatase enzymes (previously observed[11a]). In the absence of imatinib, biosensor lifetime was dynamic but overall no significant decrease in lifetime was observed (Fig. 4).
Figure 4
Subcellular Abl inhibition by imatinib
These experiments demonstrate that fluorescence lifetime shifts measured for the cell-deliverable kinase biosensor are phosphorylation-dependent, yielding dynamic information about the localization of kinase (and potentially phosphatase) activity in single living cells. This could make it possible to examine heterogeneous mixtures of cells to dissect subsets of signaling phenotypes and responses to inhibitors. This approach should also be generalizable to other kinase substrates and fluorophores, enabling the future possibility of analyzing more than one kinase-targeted FLIM biosensor at a time. Currently, we are developing other kinase substrate biosensors (e.g. for the Syk kinase[19]) to expand the application of this strategy and achieve simultaneous detection of multiple kinase activities in situ.
48 citations
TL;DR: Several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks are focused on.
Abstract: To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks.
42 citations
Cites background from "FRET-based activity biosensors to p..."
...…a NLS) or a component of a signaling complex (e.g., a scaffold protein) (Zhang et al., 2001; Kunkel and Newton, 2014), are able to monitor real-time changes in the activity profiles of specific pools of a given kinase or phosphatase in living cells (Kunkel and Newton, 2009; Gao and Zhang, 2010)....
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References
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TL;DR: It is now becoming clear that lipid micro-environments on the cell surface — known as lipid rafts — also take part in this process of signalling transduction, where protein–protein interactions result in the activation of signalling cascades.
Abstract: Signal transduction is initiated by complex protein-protein interactions between ligands, receptors and kinases, to name only a few. It is now becoming clear that lipid micro-environments on the cell surface -- known as lipid rafts -- also take part in this process. Lipid rafts containing a given set of proteins can change their size and composition in response to intra- or extracellular stimuli. This favours specific protein-protein interactions, resulting in the activation of signalling cascades.
6,080 citations
TL;DR: The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.
Abstract: Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.
5,381 citations
TL;DR: A key role of c-SRC in cancer seems to be to promote invasion and motility, functions that might contribute to tumour progression.
Abstract: The c-SRC non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been linked to the development of cancer and progression to distant metastases. These observations have led to the recent targeting of c-SRC for the development of anticancer therapeutics, which show promise as a new avenue for cancer treatment. Despite this, however, the precise functions of c-SRC in cancer remain unclear. In addition to increasing cell proliferation, a key role of c-SRC in cancer seems to be to promote invasion and motility, functions that might contribute to tumour progression.
1,130 citations
TL;DR: The presence of liquid-ordered microdomains in cells transforms the classical membrane fluid mosaic model of Singer and Nicholson into a more complex system, where proteins and lipid rafts diffuse laterally within a two-dimensional liquid.
Abstract: Lipid rafts are dynamic assemblies of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes but can also cluster to form larger, ordered platforms. Rafts are receiving increasing attention as devices that regulate membrane function in eukaryotic cells. In this Perspective, we briefly summarize the structure and regulation of lipid rafts before turning to their evident medical importance. Here, we will give some examples of how rafts contribute to our understanding of the pathogenesis of different diseases. For more information on rafts, the interested reader is referred to recent reviews (1, 2). Composition of lipid rafts Lipid rafts have changed our view of membrane organization. Rafts are small platforms, composed of sphingolipids and cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. These assemblies are fluid but more ordered and tightly packed than the surrounding bilayer. The difference in packing is due to the saturation of the hydrocarbon chains in raft sphingolipids and phospholipids as compared with the unsaturated state of fatty acids of phospholipids in the liquid-disordered phase (3). Thus, the presence of liquid-ordered microdomains in cells transforms the classical membrane fluid mosaic model of Singer and Nicholson into a more complex system, where proteins and lipid rafts diffuse laterally within a two-dimensional liquid. Membrane proteins are assigned to three categories: those that are mainly found in the rafts, those that are present in the liquid-disordered phase, and those that represent an intermediate state, moving in and out of rafts. Constitutive raft residents include glycophosphatidylinositol-anchored (GPI-anchored) proteins; doubly acylated proteins, such as tyrosine kinases of the Src family, Gα subunits of heterotrimeric G proteins, and endothelial nitric oxide synthase (eNOS); cholesterol-linked and palmitate-anchored proteins like Hedgehog (see Jeong and McMahon, this Perspective series, ref. 4); and transmembrane proteins, particularly palmitoylated proteins such as influenza virus hemagglutinin and β-secretase (BACE) (1). Some membrane proteins are regulated raft residents and have a weak affinity for rafts in the unliganded state. After binding to a ligand, they undergo a conformational change and/or become oligomerized. When proteins oligomerize, they increase their raft affinity (5). A peripheral membrane protein, such as a nonreceptor tyrosine kinase, can be reversibly palmitoylated and can lose its raft association after depalmitoylation (6). By these means, the partitioning of proteins in and out of rafts can be tightly regulated.
1,074 citations
TL;DR: A-kinase anchoring proteins are signal-organizing molecules that compartmentalize various enzymes that are regulated by second messengers that provide a molecular framework that orients these enzymes towards selected substrates.
Abstract: Multiprotein signalling networks create focal points of enzyme activity that disseminate the intracellular action of many hormones and neurotransmitters. Accordingly, the spatio-temporal activation of protein kinases and phosphatases is an important factor in controlling where and when phosphorylation events occur. Anchoring proteins provide a molecular framework that orients these enzymes towards selected substrates. A-kinase anchoring proteins (AKAPs) are signal-organizing molecules that compartmentalize various enzymes that are regulated by second messengers.
1,061 citations