High-Content Imaging and RNAi Screens for Investigating Kinase Network Plasticity

High-content imaging connects the information-rich method of microscopy with the systematic objective principles of software-driven analysis. Suited to automation and, therefore, considerable scale-up of study size, this approach can deliver multiparametric data over cell populations or at the level of the individual cell and has found considerable utility in reverse genetic and pharmacological screens. Here we present a method to screen small interfering RNA (siRNA) libraries allowing subsequent observation of the impact of each knockdown on two interlinked, high-content, G1-/S-phase cell cycle transition assays related to cyclin-dependent kinase (CDK) 2 activity. We show how plasticity within the network governing the activity of this kinase can be detected by combining modifier siRNAs with a siRNA library. The method uses fluorescent immunostaining of a nuclear antigen, CyclinA, following cell fixation while also preserving the fluorescence of a stably expressed fluorescent protein-tagged reporter for CDK2 activity. We provide methodology for data extraction and handling including an R-script that converts the multidimensional data into four simple binary outcomes, on which a hit-mining strategy can be built. The workflow described can in principle be adopted to yield quantitative single-cell-resolved data and mining for outcomes relating to a broad range of other similar readouts and signaling contexts

Signal duration and the time scale dependence of signal integration in biochemical pathways

Signal duration (e.g. the time scales over which an active signaling intermediate persists) is a key regulator of biological decisions in myriad contexts such as cell growth, proliferation, and developmental lineage commitments. Accompanying differences in signal duration are numerous downstream biological processes that require multiple steps of biochemical regulation. Here, we present an analysis that investigates how simple biochemical motifs that involve multiple stages of regulation can be constructed to differentially process signals that persist at different time scales. We compute the dynamic gain within these networks and resulting power spectra to better understand how biochemical networks can integrate signals at different time scales. We identify topological features of these networks that allow for different frequency dependent signal processing properties. Our studies suggest design principles for why signal duration in connection with multiple steps of downstream regulation is a ubiquitous control motif in biochemical systems.Comment: 27 pages, 4 figure

A Model for How Signal Duration Can Determine Distinct Outcomes of Gene Transcription Programs

The reason why IL-6 induces a pro-inflammatory response, while IL-10 induces an anti-inflammatory response, despite both cytokines activating the same transcription factor, STAT3, is not well understood. It is known that IL-6 induces a transient STAT3 signal and that IL-10 induces a sustained STAT3 signal due to the STAT3-induced inhibitor SOCS3's ability to bind to the IL-6R and not the IL-10R. We sought to develop a general transcriptional network that is capable of translating sustained signals into one response, while translating transient signals into a second response. The general structure of such a network is that the transcription factor STAT3 can induce both an inflammatory response and an anti-inflammatory response by inducing two different genes. The anti-inflammatory gene can bind to and inhibit the inflammatory gene's production and the inflammatory gene can bind to its own promoter and induce its own transcription in the absence of the signal. One prediction that can be made from such a network is that in SOCS3−/− mice, where IL-6 induces a sustained STAT3 signal, that IL-6 would act as an anti-inflammatory cytokine, which has indeed been observed experimentally in the literature

Prevalence of sexual dimorphism in mammalian phenotypic traits

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans

Cluster Lenses

Clusters of galaxies are the most recently assembled, massive, bound structures in the Universe. As predicted by General Relativity, given their masses, clusters strongly deform space-time in their vicinity. Clusters act as some of the most powerful gravitational lenses in the Universe. Light rays traversing through clusters from distant sources are hence deflected, and the resulting images of these distant objects therefore appear distorted and magnified. Lensing by clusters occurs in two regimes, each with unique observational signatures. The strong lensing regime is characterized by effects readily seen by eye, namely, the production of giant arcs, multiple-images, and arclets. The weak lensing regime is characterized by small deformations in the shapes of background galaxies only detectable statistically. Cluster lenses have been exploited successfully to address several important current questions in cosmology: (i) the study of the lens(es) - understanding cluster mass distributions and issues pertaining to cluster formation and evolution, as well as constraining the nature of dark matter; (ii) the study of the lensed objects - probing the properties of the background lensed galaxy population - which is statistically at higher redshifts and of lower intrinsic luminosity thus enabling the probing of galaxy formation at the earliest times right up to the Dark Ages; and (iii) the study of the geometry of the Universe - as the strength of lensing depends on the ratios of angular diameter distances between the lens, source and observer, lens deflections are sensitive to the value of cosmological parameters and offer a powerful geometric tool to probe Dark Energy. In this review, we present the basics of cluster lensing and provide a current status report of the field.Comment: About 120 pages - Published in Open Access at: http://www.springerlink.com/content/j183018170485723/ . arXiv admin note: text overlap with arXiv:astro-ph/0504478 and arXiv:1003.3674 by other author

ERK2 Suppresses Self-Renewal Capacity of Embryonic Stem Cells, but Is Not Required for Multi-Lineage Commitment

Activation of the FGF-ERK pathway is necessary for naïve mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification

Regulation of signal duration and the statistical dynamics of kinase activation by scaffold proteins

Scaffolding proteins that direct the assembly of multiple kinases into a spatially localized signaling complex are often essential for the maintenance of an appropriate biological response. Although scaffolds are widely believed to have dramatic effects on the dynamics of signal propagation, the mechanisms that underlie these consequences are not well understood. Here, Monte Carlo simulations of a model kinase cascade are used to investigate how the temporal characteristics of signaling cascades can be influenced by the presence of scaffold proteins. Specifically, we examine the effects of spatially localizing kinase components on a scaffold on signaling dynamics. The simulations indicate that a major effect that scaffolds exert on the dynamics of cell signaling is to control how the activation of protein kinases is distributed over time. Scaffolds can influence the timing of kinase activation by allowing for kinases to become activated over a broad range of times, thus allowing for signaling at both early and late times. Scaffold concentrations that result in optimal signal amplitude also result in the broadest distributions of times over which kinases are activated. These calculations provide insights into one mechanism that describes how the duration of a signal can potentially be regulated in a scaffold mediated protein kinase cascade. Our results illustrate another complexity in the broad array of control properties that emerge from the physical effects of spatially localizing components of kinase cascades on scaffold proteins.Comment: 12 pages, 6 figure

Reliability of Transcriptional Cycles and the Yeast Cell-Cycle Oscillator

A recently published transcriptional oscillator associated with the yeast cell cycle provides clues and raises questions about the mechanisms underlying autonomous cyclic processes in cells. Unlike other biological and synthetic oscillatory networks in the literature, this one does not seem to rely on a constitutive signal or positive auto-regulation, but rather to operate through stable transmission of a pulse on a slow positive feedback loop that determines its period. We construct a continuous-time Boolean model of this network, which permits the modeling of noise through small fluctuations in the timing of events, and show that it can sustain stable oscillations. Analysis of simpler network models shows how a few building blocks can be arranged to provide stability against fluctuations. Our findings suggest that the transcriptional oscillator in yeast belongs to a new class of biological oscillators

Sensing and Integration of Erk and PI3K Signals by Myc

The transcription factor Myc plays a central role in regulating cell-fate decisions, including proliferation, growth, and apoptosis. To maintain a normal cell physiology, it is critical that the control of Myc dynamics is precisely orchestrated. Recent studies suggest that such control of Myc can be achieved at the post-translational level via protein stability modulation. Myc is regulated by two Ras effector pathways: the extracellular signal-regulated kinase (Erk) and phosphatidylinositol 3-kinase (PI3K) pathways. To gain quantitative insight into Myc dynamics, we have developed a mathematical model to analyze post-translational regulation of Myc via sequential phosphorylation by Erk and PI3K. Our results suggest that Myc integrates Erk and PI3K signals to result in various cellular responses by differential stability control of Myc protein isoforms. Such signal integration confers a flexible dynamic range for the system output, governed by stability change. In addition, signal integration may require saturation of the input signals, leading to sensitive signal integration to the temporal features of the input signals, insensitive response to their amplitudes, and resistance to input fluctuations. We further propose that these characteristics of the protein stability control module in Myc may be commonly utilized in various cell types and classes of proteins

Observations of Lyα\alpha Emitters at High Redshift

In this series of lectures, I review our observational understanding of high-$z$ Ly$\alpha$ emitters (LAEs) and relevant scientific topics. Since the discovery of LAEs in the late 1990s, more than ten (one) thousand(s) of LAEs have been identified photometrically (spectroscopically) at $z\sim 0$ to $z\sim 10$. These large samples of LAEs are useful to address two major astrophysical issues, galaxy formation and cosmic reionization. Statistical studies have revealed the general picture of LAEs' physical properties: young stellar populations, remarkable luminosity function evolutions, compact morphologies, highly ionized inter-stellar media (ISM) with low metal/dust contents, low masses of dark-matter halos. Typical LAEs represent low-mass high-$z$ galaxies, high-$z$ analogs of dwarf galaxies, some of which are thought to be candidates of population III galaxies. These observational studies have also pinpointed rare bright Ly$\alpha$ sources extended over $\sim 10-100$ kpc, dubbed Ly$\alpha$ blobs, whose physical origins are under debate. LAEs are used as probes of cosmic reionization history through the Ly$\alpha$ damping wing absorption given by the neutral hydrogen of the inter-galactic medium (IGM), which complement the cosmic microwave background radiation and 21cm observations. The low-mass and highly-ionized population of LAEs can be major sources of cosmic reionization. The budget of ionizing photons for cosmic reionization has been constrained, although there remain large observational uncertainties in the parameters. Beyond galaxy formation and cosmic reionization, several new usages of LAEs for science frontiers have been suggested such as the distribution of {\sc Hi} gas in the circum-galactic medium and filaments of large-scale structures. On-going programs and future telescope projects, such as JWST, ELTs, and SKA, will push the horizons of the science frontiers.Comment: Lecture notes for `Lyman-alpha as an Astrophysical and Cosmological Tool', Saas-Fee Advanced Course 46. Verhamme, A., North, P., Cantalupo, S., & Atek, H. (eds.) --- 147 pages, 103 figures. Abstract abridged. Link to the lecture program including the video recording and ppt files : https://obswww.unige.ch/Courses/saas-fee-2016/program.cg