Prior to the evolution of membrane proteins, intrinsic membrane stability and permeability to polar solutes are essential features of a primitive cell membrane. These features are difficult to achieve simultaneously in model protocells made of either pure fatty acid or phospholipid membranes, raising the intriguing question of how the transition from fatty acid to phospholipid membranes might have occurred while continuously supporting encapsulated reactions required for genomic replication. Here, the properties of a blended membrane system composed of both oleic acid (OA), a monoacyl fatty acid, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a diacyl phospholipid are described. This hybrid vesicle system exhibits high stability to divalent cations (Mg ), while simultaneously maintaining its permeability to small charged molecules such as nucleotides and divalent ions such as Mg . This combination of features facilitates key reactions expected to occur during a transition from primitive to modern cells, including nonenzymatic RNA replication, and is also compatible with highly evolved functions such as the ribosomal translation of a protein. The observations support the hypothesis that the early transition from fatty acid to phospholipid membranes could be accomplished through intermediate states in which membranes are composed of amphiphile mixtures, and do not require protein transporters.

Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to highthroughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues. Ras | Erk | MAP kinase | cell signaling | dynamics | drug screen

Figure S1, related to Figure 1. Data processing pipeline for microscopy data of Erk activity. (A,C,E) Schematics of each component of the data processing pipeline that takes in multichannel TIFF image stacks of H2B-RFP and KTR-BFP as inputs, and returns quantitative data about single-cell Erk dynamics. (B) Images of H2B and KTR images where nuclei have been automatically identified (red circles). Nuclei are excluded that only express one of the two components (yellow arrows). (D) Filtering and processing of Erk activity trajectories. Representative trajectory of nuclear KTR fluorescence from a single keratinocyte over time (upper panel) is h-minima transformed, inverted, and subjected to peak-finding (lower panel). Erk activity pulse timing (blue triangles), pulse prominence (red vertical bars), and pulse width (pink horizontal bars) are obtained for each trajectory. (F) For quantifying well-averaged Erk activity, cytosolic regions are computed by a combination of dilation and erosion operations to define regions outside of nuclei. The ratio of the mean cytosolic and nuclear pixel intensities is used as a quantitative metric of the overall Erk activity offset at each timepoint. (G) Quantifying Erk activity offset for wells of keratinocytes stimulated with EGF demonstrates that a pulsatile Erk response can be sensitively detected for EGF concentrations as low as 50 pg/mL.

Phytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with Arabidopsis thaliana phytochrome B (AtPhyB) localizes to the bipartite APB motif of the A. thaliana PIFs (AtPIF). To address a dearth of quantitative interaction data, we construct and analyze numerous AtPIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of AtPIF3 and AtPIF6. We identify AtPIF variants of reduced size, monomeric or homodimeric state, and with AtPhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the AtPIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the AtPhyB:AtPIF interaction underpinning vital light-dependent responses in plants.

Biological organisms experience constantly changing environments, from sudden changes in physiology brought about by feeding, to the regular rising and setting of the Sun, to ecological changes over evolutionary timescales. Living organisms have evolved to thrive in this changing world but the general principles by which organisms shape and are shaped by time varying environments remain elusive. Our understanding is particularly poor in the intermediate regime with no separation of timescales, where the environment changes on the same timescale as the physiological or evolutionary response. Experiments to systematically characterize the response to dynamic environments are challenging since such environments are inherently high dimensional. This roadmap deals with the unique role played by time varying environments in biological phenomena across scales, from physiology to evolution, seeking to emphasize the commonalities and the challenges faced in this emerging area of research.