Temporal control of the integrated stress response by a stochastic molecular switch.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

ER-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly.

Flaviviruses, including dengue virus and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses-with ATL2 depletion leading to replication organelle defects, and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed following dengue virus infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.

Ultrastructural Characterization of Zika Virus Replication Factories.

A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.

Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale.

We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.

A Systematic Approach to Defining the microRNA Landscape in Metastasis.

The microRNA (miRNA) landscape changes during the progression of cancer. We defined a metastasis-associated miRNA landscape using a systematic approach. We profiled and validated miRNA and mRNA expression in a unique series of human colorectal metastasis tissues together with their matched primary tumors and corresponding normal tissues. We identified an exclusive miRNA signature that is differentially expressed in metastases. Three of these miRNAs were identified as key drivers of an EMT-regulating network acting though a number of novel targets. These targets include SIAH1, SETD2, ZEB2, and especially FOXN3, which we demonstrated for the first time as a direct transcriptional suppressor of N-cadherin. The modulation of N-cadherin expression had significant impact on migration, invasion, and metastasis in two different in vivo models. The significant deregulation of the miRNAs defining the network was confirmed in an independent patient set as well as in a database of diverse malignancies derived from more than 6,000 patients. Our data define a novel metastasis-orchestrating network based on systematic hypothesis generation from metastasis tissues.

Thermal robustness of signaling in bacterial chemotaxis.

Temperature is a global factor that affects the performance of all intracellular networks. Robustness against temperature variations is thus expected to be an essential network property, particularly in organisms without inherent temperature control. Here, we combine experimental analyses with computational modeling to investigate thermal robustness of signaling in chemotaxis of Escherichia coli, a relatively simple and well-established model for systems biology. We show that steady-state and kinetic pathway parameters that are essential for chemotactic performance are indeed temperature-compensated in the entire physiological range. Thermal robustness of steady-state pathway output is ensured at several levels by mutual compensation of temperature effects on activities of individual pathway components. Moreover, the effect of temperature on adaptation kinetics is counterbalanced by preprogrammed temperature dependence of enzyme synthesis and stability to achieve nearly optimal performance at the growth temperature. Similar compensatory mechanisms are expected to ensure thermal robustness in other systems.

Precision and kinetics of adaptation in bacterial chemotaxis.

The chemotaxis network of the bacterium Escherichia coli is perhaps the most studied model for adaptation of a signaling system to persistent stimuli. Although adaptation in this system is generally considered to be precise, there has been little effort to quantify this precision, or to understand how and when precision fails. Using a Förster resonance energy transfer-based reporter of signaling activity, we undertook a systematic study of adaptation kinetics and precision in E. coli cells expressing a single type of chemoreceptor (Tar). Quantifiable loss of precision of adaptation was observed at levels of the attractant MeAsp as low 10 µM, with pronounced differences in both kinetics and precision of adaptation between addition and removal of attractant. Quantitative modeling of the kinetic data suggests that loss of precise adaptation is due to a slowing of receptor methylation as available modification sites become scarce. Moreover, the observed kinetics of adaptation imply large cell-to-cell variation in adaptation rates-potentially providing genetically identical cells with the ability to "hedge their bets" by pursuing distinct chemotactic strategies.

Chemotactic response and adaptation dynamics in Escherichia coli.

Adaptation of the chemotaxis sensory pathway of the bacterium Escherichia coli is integral for detecting chemicals over a wide range of background concentrations, ultimately allowing cells to swim towards sources of attractant and away from repellents. Its biochemical mechanism based on methylation and demethylation of chemoreceptors has long been known. Despite the importance of adaptation for cell memory and behavior, the dynamics of adaptation are difficult to reconcile with current models of precise adaptation. Here, we follow time courses of signaling in response to concentration step changes of attractant using in vivo fluorescence resonance energy transfer measurements. Specifically, we use a condensed representation of adaptation time courses for efficient evaluation of different adaptation models. To quantitatively explain the data, we finally develop a dynamic model for signaling and adaptation based on the attractant flow in the experiment, signaling by cooperative receptor complexes, and multiple layers of feedback regulation for adaptation. We experimentally confirm the predicted effects of changing the enzyme-expression level and bypassing the negative feedback for demethylation. Our data analysis suggests significant imprecision in adaptation for large additions. Furthermore, our model predicts highly regulated, ultrafast adaptation in response to removal of attractant, which may be useful for fast reorientation of the cell and noise reduction in adaptation.

Variable sizes of Escherichia coli chemoreceptor signaling teams.

Like many sensory receptors, bacterial chemotaxis receptors form clusters. In bacteria, large-scale clusters are subdivided into signaling teams that act as 'antennas' allowing detection of ligands with remarkable sensitivity. The range of sensitivity is greatly extended by adaptation of receptors to changes in concentrations through covalent modification. However, surprisingly little is known about the sizes of receptor signaling teams. Here, we combine measurements of the signaling response, obtained from in vivo fluorescence resonance energy transfer, with the statistical method of principal component analysis, to quantify the size of signaling teams within the framework of the previously successful Monod-Wyman-Changeux model. We find that size of signaling teams increases 2- to 3-fold with receptor modification, indicating an additional, previously unrecognized level of adaptation of the chemotaxis network. This variation of signaling-team size shows that receptor cooperativity is dynamic and likely optimized for sensing noisy ligand concentrations.