The MAPK/ERK channel capacity exceeds 6 bit/hour.

Living cells utilize signaling pathways to sense, transduce, and process information. As the extracellular stimulation often has rich temporal characteristics which may govern dynamic cellular responses, it is important to quantify the rate of information flow through the signaling pathways. In this study, we used an epithelial cell line expressing a light-activatable FGF receptor and an ERK activity reporter to assess the ability of the MAPK/ERK pathway to transduce signal encoded in a sequence of pulses. By stimulating the cells with random light pulse trains, we demonstrated that the MAPK/ERK channel capacity is at least 6 bits per hour. The input reconstruction algorithm detects the light pulses with 1-min accuracy 5 min after their occurrence. The high information transmission rate may enable the pathway to coordinate multiple processes including cell movement and respond to rapidly varying stimuli such as chemoattracting gradients created by other cells.

Optogenetic actuator - ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics.

Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.

LITOS: a versatile LED illumination tool for optogenetic stimulation.

Optogenetics has become a key tool to manipulate biological processes with high spatio-temporal resolution. Recently, a number of commercial and open-source multi-well illumination devices have been developed to provide throughput in optogenetics experiments. However, available commercial devices remain expensive and lack flexibility, while open-source solutions require programming knowledge and/or include complex assembly processes. We present a LED Illumination Tool for Optogenetic Stimulation (LITOS) based on an assembled printed circuit board controlling a commercially available 32 × 64 LED matrix as illumination source. LITOS can be quickly assembled without any soldering, and includes an easy-to-use interface, accessible via a website hosted on the device itself. Complex light stimulation patterns can easily be programmed without coding expertise. LITOS can be used with different formats of multi-well plates, petri dishes, and flasks. We validated LITOS by measuring the activity of the MAPK/ERK signaling pathway in response to different dynamic light stimulation regimes using FGFR1 and Raf optogenetic actuators. LITOS can uniformly stimulate all the cells in a well and allows for flexible temporal stimulation schemes. LITOS's affordability and ease of use aims at democratizing optogenetics in any laboratory.

Spatiotemporal control of ERK pulse frequency coordinates fate decisions during mammary acinar morphogenesis.

The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the average frequency of non-periodic ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility and proliferation. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated multicellular ERK waves emerge, correlating with high and low ERK pulse frequencies in outer surviving and inner dying cells, respectively. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Acini harboring the PIK3CA H1047R mutation display increased ERK pulse frequency and inner cell survival. Thus, fate decisions during acinar morphogenesis are coordinated by different spatiotemporal modalities of ERK pulse frequency.

Collective ERK/Akt activity waves orchestrate epithelial homeostasis by driving apoptosis-induced survival.

Cell death events continuously challenge epithelial barrier function yet are crucial to eliminate old or critically damaged cells. How such apoptotic events are spatio-temporally organized to maintain epithelial homeostasis remains unclear. We observe waves of extracellular-signal-regulated kinase (ERK) and AKT serine/threonine kinase (Akt) activity pulses that originate from apoptotic cells and propagate radially to healthy surrounding cells. This requires epidermal growth factor receptor (EGFR) and matrix metalloproteinase (MMP) signaling. At the single-cell level, ERK/Akt waves act as spatial survival signals that locally protect cells in the vicinity of the epithelial injury from apoptosis for a period of 3-4 h. At the cell population level, ERK/Akt waves maintain epithelial homeostasis (EH) in response to mild or intense environmental insults. Disruption of this spatial signaling system results in the inability of a model epithelial tissue to ensure barrier function in response to environmental insults.

Developmental ERK Signaling Goes Digital.

Reporting in Developmental Cell, de la Cova et al. (2017) present a biosensor to measure ERK activity dynamics in C. elegans larvae. They find that fate decision signaling involves frequency-modulated, digital ERK activity pulses. These findings may explain how graded morphogen signals are converted into precise and robust cell fate patterns.