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.

CODEX, a neural network approach to explore signaling dynamics landscapes.

Current studies of cell signaling dynamics that use live cell fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional trajectories. Typically, the extraction of relevant information from time series data relies on predefined, human-interpretable features. Without a priori knowledge of the system, the predefined features may fail to cover the entire spectrum of dynamics. Here we present CODEX, a data-driven approach based on convolutional neural networks (CNNs) that identifies patterns in time series. It does not require a priori information about the biological system and the insights into the data are built through explanations of the CNNs' predictions. CODEX provides several views of the data: visualization of all the single-cell trajectories in a low-dimensional space, identification of prototypic trajectories, and extraction of distinctive motifs. We demonstrate how CODEX can provide new insights into ERK and Akt signaling in response to various growth factors, and we recapitulate findings in p53 and TGFß-SMAD2 signaling.

Irreversible Activation of Rho-activated Kinases Resulted from Evolution of Proteolytic Sites within Disordered Regions in Coiled-coil Domain.

Activation of Rho-associated protein kinase 1 (ROCK1) and myotonic dystrophy kinase-related CDC42-binding kinase alpha (MRCKα) by caspases during apoptosis in vertebrates represents a prototypical example of co-option of kinases by proteases. How caspases acquired the ability to control these proteins during evolution of vertebrates is still unknown. Here, we report a phylogenetic and molecular study on the acquisition of caspase-cleavage sites in the family of Rho-activated kinases (RaKs). We demonstrate that the acquisition of such sites has more frequently occurred in identifiable intrinsically disordered regions (IDRs) within or flanking the coiled-coil domain. Thanks to computational identification of IDRs in protein sequences of different organisms, we predicted and validated the independent evolution of two caspase-cleavage sites in ROCK of arthropods and the loss of one of the MRCKα caspase-cleavage sites in ray-finned fishes. In conclusion, we shed light on the propensity of RaKs to evolve novel proteolytic sites, causing kinase activation and uniform subcellular distribution.

Death for life: a path from apoptotic signaling to tissue-scale effects of apoptotic epithelial extrusion.

Apoptosis plays a crucial role in clearing old or critically compromised cells, and actively maintains epithelial homeostasis and epithelial morphogenesis during embryo development. But how is the apoptotic signaling pathway able to orchestrate such complex and dynamic multi-cellular morphological events at the tissue scale? In this review we collected the most updated knowledge regarding how apoptosis controls different cytoskeletal components. We describe how apoptosis can control epithelial homeostasis though epithelial extrusion, a highly orchestrated process based on high- order actomyosin structures and on the coordination between the apoptotic and the neighboring cells. Finally, we describe how the synergy among forces generated by multiple apoptotic cells can shape epithelia in embryo development.

PDK1: At the crossroad of cancer signaling pathways.

Rational target therapy of cancer would benefit from the identification of new targets that can be easily inhibited by small molecules. An increasing amount of evidence hints at 3-phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) as an intriguing and underexplored target for cancer therapy. Several reports show that PDK1 expression is dysregulated in multiple cancer types. Furthermore PDK1 is implicated in signaling pathways frequently altered in cancer, such as PI3K/Akt, Ras/MAPK and Myc. PDK1 targeting has been proven to be effective in experimental models harboring alterations of these pathways. In this paper we review PDK1 main biochemical mechanisms, its alterations in cancer and interactions with relevant cancer pathways. A potential role of PDK1 in tumor microenvironment is also discussed.

PDK1 regulates focal adhesion disassembly by modulating endocytosis of αvß3 integrin.

Non-amoeboid cell migration is characterised by dynamic competition among multiple protrusions to establish new adhesion sites at the cell's leading edge. However, the mechanisms that regulate the decision to disassemble or to grow nascent adhesions are not fully understood. Here we show that, in endothelial cells, 3-phosphoinositide-dependent protein kinase 1 (PDK1) promotes focal adhesion (FA) turnover by controlling endocytosis of integrin αvß3 in a PI3K-dependent manner. We demonstrate that PDK1 binds and phosphorylates integrin αvß3. Downregulation of PDK1 increases FA size and slows down their disassembly. This process requires both PDK1 kinase activity and PI3K activation but does not involve Akt. Moreover, PDK1 silencing stabilises FA in membrane protrusions decreasing migration of endothelial cells on vitronectin. These results indicate that modulation of integrin endocytosis by PDK1 hampers endothelial cell adhesion and migration on extracellular matrix, thus unveiling a novel role for this kinase.

PDK1: A signaling hub for cell migration and tumor invasion.

The ability of cells to migrate is essential for different physiological processes including embryonic development, angiogenesis, tissue repair and immune response. In the context of cancer such abilities acquire dramatic implications, as they are exploited by tumor cells to invade neighboring or distant healthy tissues. 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) is an ancient serine-threonine kinase belonging to AGC kinase family. An increasing amount of data points at a pivotal role for PDK1 in the regulation of cell migration. PDK1 is a transducer of PI3K signaling and activates multiple downstream effectors, thereby representing an essential hub coordinating signals coming from extracellular cues to the cytoskeletal machinery, the final executor of cell movement. Akt, PAK1, ß3 integrin, ROCK1, MRCKα and PLCγ1 are, according to the literature, the signaling transducers through which PDK1 regulates cell migration. In addition, PDK1 contributes to tumor cell invasion by regulating invadopodia formation and both amoeboid and collective cancer cell invasion. This and other pieces of evidence, such as its reported overexpression across several tumor types, corroborate a PDK1 role tumor aggressiveness. Altogether, these findings indicate the possibility to rationally target PDK1 in human tumors in order to counteract cancer cell dissemination in the organism.

Modeling human tumor angiogenesis in a three-dimensional culture system.

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.

The mitogen-activated protein kinase network, wired to dynamically function at multiple scales.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling network is a key transducer of signals from various receptors, including receptor tyrosine kinases (RTKs). It controls cell-cycle entry, survival, motility, differentiation, as well as other fates. After four decades of studying this pathway with biochemical methods, the use of fluorescent biosensors has revealed dynamic behaviors such as ERK pulsing, oscillations, and amplitude-modulated activity. Different RTKs equip the MAPK network with specific feedback mechanisms to encode these different ERK dynamics, which are then subsequently decoded into cytoskeletal events and transcriptional programs, actuating cellular fates. Recently, collective ERK wave behaviors have been observed in multiple systems to coordinate cytoskeletal dynamics with fate decisions within cell collectives. This emphasizes that a correct understanding of this pathway requires studying it at multiple scales.

Transformer-based spatial-temporal detection of apoptotic cell death in live-cell imaging.

Intravital microscopy has revolutionized live-cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy timelapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy timelapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in mice, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85ß.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85ß). Down-regulation of miR-126 and over-expression of p85ß in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

Automatic detection of spatio-temporal signaling patterns in cell collectives.

Increasing experimental evidence points to the physiological importance of space-time correlations in signaling of cell collectives. From wound healing to epithelial homeostasis to morphogenesis, coordinated activation of biomolecules between cells allows the collectives to perform more complex tasks and to better tackle environmental challenges. To capture this information exchange and to advance new theories of emergent phenomena, we created ARCOS, a computational method to detect and quantify collective signaling. We demonstrate ARCOS on cell and organism collectives with space-time correlations on different scales in 2D and 3D. We made a new observation that oncogenic mutations in the MAPK/ERK and PIK3CA/Akt pathways of MCF10A epithelial cells hyperstimulate intercellular ERK activity waves that are largely dependent on matrix metalloproteinase intercellular signaling. ARCOS is open-source and available as R and Python packages. It also includes a plugin for the napari image viewer to interactively quantify collective phenomena without prior programming experience.

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.

Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints.

The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.

Colorectal cancer residual disease at maximal response to EGFR blockade displays a druggable Paneth cell-like phenotype.

Blockade of epidermal growth factor receptor (EGFR) causes tumor regression in some patients with metastatic colorectal cancer (mCRC). However, residual disease reservoirs typically remain even after maximal response to therapy, leading to relapse. Using patient-derived xenografts (PDXs), we observed that mCRC cells surviving EGFR inhibition exhibited gene expression patterns similar to those of a quiescent subpopulation of normal intestinal secretory precursors with Paneth cell characteristics. Compared with untreated tumors, these pseudodifferentiated tumor remnants had reduced expression of genes encoding EGFR-activating ligands, enhanced activity of human epidermal growth factor receptor 2 (HER2) and HER3, and persistent signaling along the phosphatidylinositol 3-kinase (PI3K) pathway. Clinically, properties of residual disease cells from the PDX models were detected in lingering tumors of responsive patients and in tumors of individuals who had experienced early recurrence. Mechanistically, residual tumor reprogramming after EGFR neutralization was mediated by inactivation of Yes-associated protein (YAP), a master regulator of intestinal epithelium recovery from injury. In preclinical trials, Pan-HER antibodies minimized residual disease, blunted PI3K signaling, and induced long-term tumor control after treatment discontinuation. We found that tolerance to EGFR inhibition is characterized by inactivation of an intrinsic lineage program that drives both regenerative signaling during intestinal repair and EGFR-dependent tumorigenesis. Thus, our results shed light on CRC lineage plasticity as an adaptive escape mechanism from EGFR-targeted therapy and suggest opportunities to preemptively target residual disease.

Developmental Erk Signaling Illuminated.

How a small number of signaling pathways can be re-used in distinct embryonic contexts to control different fates remains unclear. In this issue of Developmental Cell, Johnson and Toettcher (2019) use optogenetic approaches to explore how different dynamic ERK signaling states control specific developmental fates in the Drosophila embryo.

PI3K/mTOR inhibition promotes the regression of experimental vascular malformations driven by PIK3CA-activating mutations.

Somatic activating mutations within the PIK3CA gene have been recently detected in sporadic lymphatic and venous malformations, and in vascular malformations (VM) associated to overgrowth syndromes, such as CLOVES and Klippel-Trenaunay syndrome. Although VM are often limited to specific tissue areas and can be well treated, in extended or recurrent lesions novel therapeutic approaches are needed. We generated a mouse model of VM by local expression of PIK3CA-activating mutation in endothelial cells. PIK3CA-driven lesions are characterized by large areas of hemorrhage, hyperplastic vessels, infiltrates of inflammatory cells, and elevated endothelial cell density. Such vascular lesions are ameliorated by administration of dual PI3K/mTOR inhibitor, BEZ235, and mTOR inhibitor, Everolimus. Unexpectedly, the expression of PIK3CA-activating mutations in human endothelial cells results in both increased proliferation rates and senescence. Moreover, active forms of PIK3CA strongly promote the angiogenic sprouting. Treatment with PI3K/mTOR inhibitors restores normal endothelial cell proliferation rate and reduces the amount of senescent cells, whereas treatment with Akt inhibitor is less effective. Our findings reveal that PIK3CA mutations have a key role in the pathogenesis of VM and PIK3CA-driven experimental lesions can be effectively treated by PI3K/mTOR inhibitors.