CDK2 activity crosstalk on the ERK kinase translocation reporter can be resolved computationally.

The mitogen-activated protein kinase (MAPK) pathway integrates growth factor signaling through extracellular signal-regulated kinase (ERK) to control cell proliferation. To study ERK dynamics, many researchers use an ERK activity kinase translocation reporter (KTR). Our study reveals that this ERK KTR also partially senses cyclin-dependent kinase 2 (CDK2) activity, making it appear as if ERK activity rises as cells progress through the cell cycle.

CDK4/6 activity is required during G arrest to prevent stress-induced endoreplication.

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G-arrested cells to prevent cell cycle exit after the S phase.

Oncogenic Kras induces spatiotemporally specific tissue deformation through converting pulsatile into sustained ERK activation.

Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue.

Sustained ERK signaling promotes G2 cell cycle exit and primes cells for whole-genome duplication.

Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling.

Organ function is preserved despite reorganization of niche architecture in the hair follicle.

The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture.

Systematic analysis of the MAPK signaling network reveals MAP3K-driven control of cell fate.

The classic network of mitogen-activated protein kinases (MAPKs) is highly interconnected and controls a diverse array of biological processes. In multicellular eukaryotes, the MAPKs ERK, JNK, and p38 control opposing cell behaviors but are often activated simultaneously, raising questions about how input-output specificity is achieved. Here, we use multiplexed MAPK activity biosensors to investigate how cell fate control emerges from the connectivity and dynamics of the MAPK network.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca and activate transcriptional programs important for exercise and immunity.

3D time-lapse microscopy paired with endpoint lineage analysis in mouse blastocysts.

Determining how signaling dynamics relate to gene expression and cell fate is essential to understanding multicellular development. We present a unified live imaging and lineage analysis method that allows integrated analysis of both techniques in the same mouse embryos. This protocol describes the embryo isolation, confocal imaging, immunofluorescence, and alignment required to connect time-lapse and endpoint measurements.

Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear.

Cell-Cycle-Dependent ERK Signaling Dynamics Direct Fate Specification in the Mammalian Preimplantation Embryo.

Despite the noisy nature of single cells, multicellular organisms robustly generate different cell types from one zygote. This process involves dynamic cross regulation between signaling and gene expression that is difficult to capture with fixed-cell approaches. To study signaling dynamics and fate specification during preimplantation development, we generated a transgenic mouse expressing the ERK kinase translocation reporter and measured ERK activity in single cells of live embryos.

MAPK activity dynamics regulate non-cell autonomous effects of oncogene expression.

A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e.

Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate.

Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction.

Stress-mediated exit to quiescence restricted by increasing persistence in CDK4/6 activation.

Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood.

NF-κB signaling dynamics is controlled by a dose-sensing autoregulatory loop.

Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network.

Live-cell measurements of kinase activity in single cells using translocation reporters.

Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization.

Temporal Control of Mammalian Cortical Neurogenesis by mA Methylation.

N-methyladenosine (mA), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether mA regulates mammalian brain development is unknown. Here, we show that mA depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages.

A Real-Time Biosensor for ERK Activity Reveals Signaling Dynamics during C. elegans Cell Fate Specification.

Kinase translocation reporters (KTRs) are genetically encoded fluorescent activity sensors that convert kinase activity into a nucleocytoplasmic shuttling equilibrium for visualizing single-cell signaling dynamics. Here, we adapt the first-generation KTR for extracellular signal-regulated kinase (ERK) to allow easy implementation in vivo. This sensor, "ERK-nKTR," allows quantitative and qualitative assessment of ERK activity by analysis of individual nuclei and faithfully reports ERK activity during development and neural function in diverse cell contexts in Caenorhabditis elegans.

Parallel feedback loops control the basal activity of the HOG MAPK signaling cascade.

Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation.

High-sensitivity measurements of multiple kinase activities in live single cells.

Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells.

Accelerated discovery via a whole-cell model.

To test the promise of whole-cell modeling to facilitate scientific inquiry, we compared growth rates simulated in a whole-cell model with experimental measurements for all viable single-gene disruption Mycoplasma genitalium strains. Discrepancies between simulations and experiments led to predictions about kinetic parameters of specific enzymes that we subsequently validated. These findings represent, to our knowledge, the first application of whole-cell modeling to accelerate biological discovery.

The Hog1 stress-activated protein kinase targets nucleoporins to control mRNA export upon stress.

The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation.

Distributed biological computation with multicellular engineered networks.

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells.

The HOG pathway dictates the short-term translational response after hyperosmotic shock.

Cellular responses to environmental changes occur on different levels. We investigated the translational response of yeast cells after mild hyperosmotic shock by isolating mRNA associated with multiple ribosomes (polysomes) followed by array analysis. Globally, recruitment of preexisting mRNAs to ribosomes (translational response) is faster than the transcriptional response.