Elucidating Extracellular Signal-Regulated Kinase (ERK) Dynamics With FRET-based Biosensors

Proper cell functions are dependent on the precise regulation of signal transduction pathways to relay specific external environmental cues into the cell for processing and yielding a specific response. Dysregulation of signaling function or localization is a hallmark of numerous diseases including neurodegenerative, autoimmune, and immunological disorders and most human cancers. Given that signaling pathways are large networks of nodes capable of interacting in complex manners, a notable question that arises for many studying the intricacies of signaling regulation is: “How is signaling tightly controlled in different subcellular compartments?” Here we examine and elucidate mechanisms that begin to answer this fundamental question in the context of EGF signaling. Conventional techniques have been successful in investigating the functions of individual components and in mapping interactions between signaling components. However, discrete time snapshots of activity in a population of cells or the analysis of single components of signaling pathways outside of their natural biological context leave much information about spatial regulation, cell-cell variability, and time-resolved dynamical information unrevealed. Addressing these limitations, FRET-based biosensors provide a powerful means for dissecting the complexities of signaling pathway components in real-time with single cell resolution in their native live biological context. Utilizing engineered and optimized ERK Activity Reporters (EKARs), we examine ERK activity dynamics in the context of epidermal growth factor (EGF) signaling in PC12 cells, a cell line historical to questions related to signal specificity. We reveal distinct ERK activity dynamics in different subcellular compartments, the presence of switch-like activation of plasma membrane (PM) ERK, and elucidate mechanisms involved in controlling the switch-like activation of PM ERK. Next, we examine how the signaling network topology we uncovered at the PM is coupled (or uncoupled) to cytoplasmic ERK activity. Lastly, we show distinct functional roles for the different pools of ERK activity

FRET-BASED BIOSENSORS TO ELUCIDATE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) DYNAMICS

Proper cell functions are dependent on the precise regulation of transduction events to relay external environmental cues into the cell for processing. The propagation and regulation of the signal involves a diverse array of enzymes and molecules with distinct functions and differences in spatio-temporal regulation. Further understanding cell signaling dynamics will continue to provide a knowledge basis for developing disease therapies. While an active field of research, a great deal of information has remained elusive with the previously used tools to monitor such pathways either through discrete snapshots or analyses of single components of signaling pathways outside of their natural biological context. To address these limitations, FRET-based biosensors provide a powerful means to dissect the complexities of signaling pathways in a real-time manner in live cells. The first objective is to optimize the previously developed ERK Activity Reporter, EKAR, by examining the dependence of its dynamic range on fluorophore orientation. To alter relative fluorophore orientations, variations of EKAR were developed with reversed orders of fluorescent proteins. Reversing the order of a previously optimized yellow-cyan EKAR led to a cyan-yellow EKAR with an improved dynamic range. Moreover, a closer examination of the previously optimized EeVee-linker, which was believed to render FRET-based biosensors solely distance-dependent, reveals that the FRET efficiency is likely dependent on FP orientation in EeVee-linker based biosensors. This conclusion for EKAR can likely be extended for further improvements of other FRET-based biosensors with the EeVee linker. The second objective is to expand the versatility of the EKAR biosensor for co-imaging capabilities, which allow for more precise correlations between multiple activity readouts on a single-cell level. Utilizing EKAR color variants, a co-imaging strategy was employed to allow for imaging two sensors with spectrally distinct FRET-donor fluorescent proteins (FP) and a common FRET-acceptor FP. This strategy allowed for simultaneously tracking ERK activity in multiple subcellular compartments, monitoring both ERK and Protein kinase A (PKA) on a single-cell level, and for the development of a novel single-chain biosensor capable of monitoring ERK and Ras activity in the plasma membrane. Continued investigations will provide powerful tools to elucidate signaling dynamics with stronger precision

A molecular clock controls periodically driven cell migration in confined spaces

AbstractNavigation through dense, physically confining extracellular matrix is common in invasive cell spread and tissue re-organization, but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTP-ase RhoA, dependent on the oscillatory changes in the activity and abundance of the RhoA Guanine Exchange Factor, GEF-H1, triggered by a persistent increase in the intracellular Ca2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with the frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments.</jats:p

Signaling Diversity Enabled by Rap1-Regulated Plasma Membrane ERK with Distinct Temporal Dynamics

AbstractA variety of different signals induce specific responses through a common, ERK-dependent kinase cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of FRET-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK enzymatic activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytopolasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.</jats:p

Signaling diversity enabled by Rap1-regulated plasma membrane ERK with distinct temporal dynamics.

A variety of different signals induce specific responses through a common, extracellular-signal regulated kinase (ERK)-dependent cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubtion of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we have expanded the toolbox of Förster Resonance Energy Transfer (FRET)-based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytoplasm and nucleus. Furthermore, EGF-induced plasma membrane ERK activity involves Rap1, a noncanonical activator, and controls cell morphology and EGF-induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes