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  • 1
    Keywords: IN-VIVO ; MAMMALIAN-CELLS ; LIVING CELLS ; EMBRYONIC STEM-CELLS ; LYSINE-9 METHYLATION ; HP1 PROTEINS ; DNA-METHYLATION ; HISTONE H3 ; CHROMATIN-REMODELING COMPLEXES ; NUCLEOSOME MODIFICATION
    Abstract: The cell establishes heritable patterns of active and silenced chromatin via interacting factors that set, remove, and read epigenetic marks. To understand how the underlying networks operate, we have dissected transcriptional silencing in pericentric heterochromatin (PCH) of mouse fibroblasts. We assembled a quantitative map for the abundance and interactions of 16 factors related to PCH in living cells and found that stably bound complexes of the histone methyltransferase SUV39H1/2 demarcate the PCH state. From the experimental data, we developed a predictive mathematical model that explains how chromatin-bound SUV39H1/2 complexes act as nucleation sites and propagate a spatially confined PCH domain with elevated histone H3 lysine 9 trimethylation levels via chromatin dynamics. This "nucleation and looping" mechanism is particularly robust toward transient perturbations and stably maintains the PCH state. These features make it an attractive model for establishing functional epigenetic domains throughout the genome based on the localized immobilization of chromatin-modifying enzymes.
    Type of Publication: Journal article published
    PubMed ID: 25134515
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  • 2
  • 3
    Keywords: CELL ; Germany ; KINASE ; PATHWAY ; PATHWAYS ; SYSTEMS ; SITE ; SITES ; DISTINCT ; PROTEIN ; TIME ; ACTIVATION ; RESPONSES ; MECHANISM ; BINDING ; PHOSPHORYLATION ; TARGET ; PATTERNS ; SIGNAL-TRANSDUCTION ; ULTRASENSITIVITY ; AFFINITY ; signaling ; SINGLE ; INCREASE ; PHOSPHORYLATION SITES ; ENZYME ; analysis ; PHOSPHATASE ; PREDICT ; INCREASES ; DUAL PHOSPHORYLATION ; INTERCONVERTIBLE ENZYME CASCADES ; METABOLIC-REGULATION ; MULTIPLE PHOSPHORYLATION ; multisite phosphorylation ; order of phosphate processing ; stimulus-response relationship ; TRANSCRIPTION FACTOR NFAT1 ; transition time
    Abstract: Multisite protein phosphorylation is a common regulatory mechanism in cell signaling, and dramatically increases the possibilities for protein-protein interactions, conformational regulation, and phosphorylation pathways. However, there is at present no comprehensive picture of how these factors shape the response of a protein's phosphorylation state to changes in kinase and phosphatase activities. Here we provide a mathematical theory for the regulation of multisite protein phosphorylation based on the mechanistic description of elementary binding and catalytic steps. Explicit solutions for the steady-state response curves and characteristic (de)phosphorylation times have been obtained in special cases. The order of phosphate processing and the characteristics of protein-protein interactions turn out to be of overriding importance for both sensitivity and speed of response. Random phosphate processing gives rise to shallow response curves, favoring intermediate phosphorylation states of the target, and rapid kinetics. Sequential processing is characterized by steeper response curves and slower kinetics. We show systematically how qualitative differences in target phosphorylation - including graded, switch-like and bistable responses - are determined by the relative concentrations of enzyme and target as well as the enzyme-target affinities. In addition to collective effects of several phosphorylation sites, our analysis predicts that distinct phosphorylation patterns can be finely tuned by a single kinase. Taken together, this study suggests a versatile regulation of protein activation by the combined effect of structural, kinetic and thermodynamic aspects of multisite phosphorylation
    Type of Publication: Journal article published
    PubMed ID: 17257173
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  • 4
    Keywords: EXPRESSION ; Germany ; KINASE ; MODEL ; MODELS ; ENZYMES ; GENE ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; METABOLISM ; TRANSDUCTION ; RESPONSES ; MECHANISM ; mechanisms ; PHOSPHORYLATION ; signal transduction ; SIGNAL ; TARGET ; gene expression ; NUMBER ; SIGNAL-TRANSDUCTION ; PARAMETERS ; CALCIUM ; sensitivity ; specificity ; PROTEIN-PHOSPHORYLATION ; signaling ; ORDER ; molecular ; RE ; ENZYME ; USA ; SIGNALS ; modeling ; SHAPE ; CAM KINASE-II ; COMPLEX CA2+ SIGNALS ; CYTOSOLIC CALCIUM ; GLYCOGEN-PHOSPHORYLASE ; MATHEMATICAL-ANALYSIS ; MINIMAL MODEL ; PHOSPHOLIPASE C-DELTA-1 ; SPIKE FREQUENCY
    Abstract: Experimental studies have demonstrated that Ca2+-regulated proteins are sensitive to the frequency of Ca2+ oscillations, and several mathematical models for specific proteins have provided insight into the mechanisms involved. Because of the large number of Ca2+-regulated proteins in signal transduction, metabolism and gene expression, it is desirable to establish in general terms which molecular properties shape the response to oscillatory Ca2+ signals. Here we address this question by analyzing in detail a model of a prototypical Ca2+-decoding module, consisting of a target protein whose activity is controlled by a Ca2+-activated kinase and the counteracting phosphatase. We show that this module can decode the frequency of Ca2+ oscillations, at constant average Ca2+ signal, provided that the Ca2+ spikes are narrow and the oscillation frequency is sufficiently low-of the order of the phosphatase rate constant or below. Moreover, Ca2+ oscillations activate the target more efficiently than a constant signal when Ca2+ is bound cooperatively and with low affinity. Thus, the rate constants and the Ca2+ affinities of the target-modifying enzymes can be tuned in such a way that the module responds optimally to Ca2+ spikes of a certain amplitude and frequency. Frequency sensitivity is further enhanced when the limited duration of the external stimulus driving Ca2+ signaling is accounted for. Thus, our study identifies molecular parameters that may be involved in establishing the specificity of cellular responses downstream of Ca2+ oscillations
    Type of Publication: Journal article published
    PubMed ID: 17921221
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  • 5
    Keywords: CELL ; Germany ; MODEL ; CRASSA ; GENE ; PROTEIN ; transcription ; ACCUMULATION ; TIME ; COMPLEX ; COMPLEXES ; TRANSCRIPTION FACTOR ; BIOLOGY ; CYCLE ; PHOSPHORYLATION ; FREQUENCIES ; Drosophila ; genetics ; DISPLAY ; LOCALIZATION ; NUCLEUS ; KINETICS ; REGULATOR ; PROTEIN-PHOSPHORYLATION ; SCALE ; LEVEL ; USA ; EXPORT ; STEADY-STATE ; CIRCADIAN CLOCK ; Genetic ; Developmental ; TRANSCRIPTION-FACTOR ; ENTRAINMENT ; EPSILON ; FRQ ; GENE-FREQUENCY ; INTERLOCKED FEEDBACK LOOPS ; OUTPUT ; RHYTHMS ; subcellular shuttling
    Abstract: The Neurospora clock protein FREQUENCY (FRQ) is an essential regulator of the circadian transcription factor WHITE COLLAR COMPLEX (WCC). In the course of a circadian period, the subcellular distribution of FRQ shifts from mainly nuclear to mainly cytosolic. This shift is crucial for coordinating the negative and positive limbs of the clock. We show that the subcellular redistribution of FRQ on a circadian time scale is governed by rapid, noncircadian cycles of nuclear import and export. The rate of nuclear import of newly synthesized FRQ is progressively reduced in a phosphorylation-dependent manner, leading to an increase in the steady-state level of cytoplasmic FRQ. The long-period frq(7) mutant displays reduced kinetics of FRQ(7) protein phosphorylation and a prolonged accumulation in the nucleus. We present a mathematical model that describes the cytoplasmic accumulation of wild-type and mutant FRQ on a circadian time scale on the basis of frequency-modulated rapid nucleocytoplasmic shuttling cycles
    Type of Publication: Journal article published
    PubMed ID: 19759264
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  • 6
    Keywords: Germany ; MODEL ; MODELS ; SITES ; PROTEIN ; MECHANISM ; mechanisms ; BIOLOGY ; PHOSPHORYLATION ; DNA-REPLICATION ; KINETICS ; sensitivity ; specificity ; ULTRASENSITIVITY ; TYROSINE PHOSPHORYLATION ; PROTEIN-PHOSPHORYLATION ; molecular biology ; review ; regulation ; SRC FAMILY KINASES ; PHOSPHORYLATION SITES ; PROCESSIVITY ; USA ; MULTIPLE PHOSPHORYLATION ; TRANSCRIPTION FACTOR NFAT1 ; WELL ; mathematical models ; DROSOPHILA PERIOD PROTEIN ; DUAL-KINASE MECHANISM ; enzyme processivity ; kinetic proofreading ; order of phospho-site processing ; PROCESSIVE PHOSPHORYLATION ; SIGNAL-TRANSDUCTION CASCADES ; SPLICING FACTOR ASF/SF2
    Abstract: Multisite phosphorylation is an important mechanism for fine-tuned regulation of protein function. Mathematical models developed over recent years have contributed to elucidation of the functional consequences of a variety of molecular mechanisms involved in processing of the phosphorylation sites. Here we review the results of such models, together with salient experimental findings on multisite protein phosphorylation. We discuss how molecular mechanisms that can be distinguished with respect to the order and processivity of phosphorylation, as well as other factors, regulate changes in the sensitivity and kinetics of the response, the synchronization of molecular events, signalling specificity, and other functional implications
    Type of Publication: Journal article published
    PubMed ID: 19438722
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  • 7
    Keywords: ACTIVATION ; T-CELLS ; BIOLOGY ; RECOGNITION ; sensitivity ; DOMAINS ; STOICHIOMETRY ; BEAD ARRAY ; CELL ANTIGEN-RECEPTOR ; TYROSINE KINASE ZAP-70
    Abstract: Background: To understand complex biological signalling mechanisms, mathematical modelling of signal transduction pathways has been applied successfully in last few years. However, precise quantitative measurements of signal transduction events such as activation-dependent phosphorylation of proteins, remains one bottleneck to this success. Methodology/Principal Findings: We use multi-colour immunoprecipitation measured by flow cytometry (IP-FCM) for studying signal transduction events to unrivalled precision. In this method, antibody-coupled latex beads capture the protein of interest from cellular lysates and are then stained with differently fluorescent-labelled antibodies to quantify the amount of the immunoprecipitated protein, of an interaction partner and of phosphorylation sites. The fluorescence signals are measured by FCM. Combining this procedure with beads containing defined amounts of a fluorophore allows retrieving absolute numbers of stained proteins, and not only relative values. Using IP-FCM we derived multidimensional data on the membrane-proximal T-cell antigen receptor (TCR-CD3) signalling network, including the recruitment of the kinase ZAP70 to the TCR-CD3 and subsequent ZAP70 activation by phosphorylation in the murine T-cell hybridoma and primary murine T cells. Counter-intuitively, these data showed that cell stimulation by pervanadate led to a transient decrease of the phospho-ZAP70/ZAP70 ratio at the TCR. A mechanistic mathematical model of the underlying processes demonstrated that an initial massive recruitment of non-phosphorylated ZAP70 was responsible for this behaviour. Further, the model predicted a temporal order of multisite phosphorylation of ZAP70 (with Y319 phosphorylation preceding phosphorylation at Y493) that we subsequently verified experimentally. Conclusions/Significance: The quantitative data sets generated by IP-FCM are one order of magnitude more precise than Western blot data. This accuracy allowed us to gain unequalled insight into the dynamics of the TCR-CD3-ZAP70 signalling network
    Type of Publication: Journal article published
    PubMed ID: 21829558
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  • 8
    Keywords: CELLS ; EXPRESSION ; imaging ; GENE ; GENE-EXPRESSION ; PROTEINS ; ACTIVATION ; DNA ; recombination ; INDUCTION ; RECOGNITION ; gene expression ; VARIABILITY ; VIRUS-INFECTION ; SINGLE-CELL ; RIG-I ; BACTERIAL ARTIFICIAL CHROMOSOMES ; BETA GENE-EXPRESSION ; interferon regulatory factor-7 ; live-cell microscopy ; multi-scale modelling
    Abstract: The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-beta expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-beta expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-kappa B and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
    Type of Publication: Journal article published
    PubMed ID: 22617958
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  • 9
    Keywords: DIFFERENTIATION ; CUTTING EDGE ; TRANSCRIPTION FACTOR ; MEMORY ; SUBSETS ; EFFECTOR ; PRECURSOR ; EXPANSION ; STEM ; DIVISION
    Abstract: A core feature of protective T cell responses to infection is the robust expansion and diversification of naive antigen-specific T cell populations into short-lived effector and long-lived memory subsets. By means of in vivo fate mapping, we found a striking variability of immune responses derived from individual CD8(+) T cells and show that robust acute and recall immunity requires the initial recruitment of multiple precursors. Unbiased mathematical modeling identifies the random integration of multiple differentiation and division events as the driving force behind this variability. Within this probabilistic framework, cell fate is specified along a linear developmental path that progresses from slowly proliferating long-lived to rapidly expanding short-lived subsets. These data provide insights into how complex biological systems implement stochastic processes to guarantee robust outcomes.
    Type of Publication: Journal article published
    PubMed ID: 23493420
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  • 10
    Keywords: IN-VIVO ; DIFFERENTIATION ; BONE-MARROW ; EFFECTOR ; ADOPTIVE TRANSFER ; SELF-RENEWAL ; LISTERIA-MONOCYTOGENES ; COLONY-FORMING CELLS ; CD8-ALPHA(+) DENDRITIC CELLS ; SPLEEN COLONIES
    Abstract: Maintenance of immunological memory has been proposed to rely on stem-cell-like lymphocytes. However, data supporting this hypothesis are focused on the developmental potential of lymphocyte populations and are thus insufficient to establish the functional hallmarks of stemness. Here, we investigated self-renewal capacity and multipotency of individual memory lymphocytes by in vivo fate mapping of CD8(+) T cells and their descendants across three generations of serial single-cell adoptive transfer and infection-driven re-expansion. We found that immune responses derived from single naive T (Tn) cells, single primary, and single secondary central memory T (Tcm) cells reached similar size and phenotypic diversity, were subjected to comparable stochastic variation, and could ultimately reconstitute immunocompetence against an otherwise lethal infection with the bacterial pathogen Listeria monocytogenes. These observations establish that adult tissue stem cells reside within the CD62L(+) Tcm cell compartment and highlight the promising therapeutic potential of this immune cell subset.
    Type of Publication: Journal article published
    PubMed ID: 25035956
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