A redox signaling mechanism for density-dependent inhibition of cell growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.     

A flip-flop switch in polarity signaling

The Rho GTPase Cdc42 is essential for polarized growth of budding yeast. Temporal control of Cdc42 depends partly on the activity of its GTPase-activating proteins (GAPs). In this issue of Developmental Cell, Saito et al. report that Cdc42 GAP activity is regulated by the phospholipid composition of the bud-tip membrane, under control of the phospholipid flippases Lem3-Dnf1 and Lem3-Dnf2.     

Receptor clustering drives polarized assembly of ankyrin

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.     

Population extinction and quasi-stationary behavior in stochastic density-dependent structured models

Density-independent and density-dependent, stochastic and deterministic, discrete-time, structured models are formulated, analysed and numerically simulated. A special case of the deterministic, density-independent, structured model is the well-known Leslie age-structured model. The stochastic, density-independent model is a multitype branching process. A review of linear, density-independent models is given first, then nonlinear, density-dependent models are discussed. In the linear, density-independent structured models, transitions between states are independent of time and state. Population extinction is determined by the dominant eigenvalue λ of the transition matrix. If λ ≤ 1, then extinction occurs with probability one in the stochastic and deterministic models. However, if λ > 1, then the deterministic model has exponential growth, but in the stochastic model there is a positive probability of extinction which depends on the fixed point of the system of probability generating functions. The linear, density-independent, stochastic model is generalized to a nonlinear, density-dependent one. The dependence on state is in terms of a weighted total population size. It is shown for small initial population sizes that the density-dependent, stochastic model can be approximated by the density-independent, stochastic model and thus, the extinction behavior exhibited by the linear model occurs in the nonlinear model. In the deterministic models there is a unique stable equilibrium. Given the population does not go extinct, it is shown that the stochastic model has a quasi-stationary distribution with mean close to the stable equilibrium, provided the population size is sufficiently large. For small values of the population size, complete extinction can be observed in the simulations. However, the persistence time increases rapidly with the population size. This author received partial support by the National Science Foundation grant # DMS-9626417.     

Finite metapopulation models with density-dependent migration and stochastic local dynamics

The effects of small density-dependent migration on the dynamics of a metapopulation are studied in a model with stochastic local dynamics. We use a diffusion approximation to study how changes in the migration rate and habitat occupancy affect the rates of local colonization and extinction. If the emigration rate increases or if the immigration rate decreases with local population size, a positive expected rate of change in habitat occupancy is found for a greater range of habitat occupancies than when the migration is density-independent. In contrast, the reverse patterns of density dependence in respective emigration and immigration reduce the range of habitat occupancies where the metapopulation will be viable. This occurs because density-dependent migration strongly influences both the establishment and rescue effects in the local dynamics of metapopulations.     

ER signaling regulation drives the switch between autophagy and apoptosis in NRK-52E cells exposed to cisplatin

Cisplatin (cisPt) use in chemotherapy is limited by the occurrence of a severe nephrotoxicity. Both autophagy and apoptosis seem to contribute in kidney response to cisPt, however their cross-talk is still controversial, since the role played by autophagy (cytoprotective or harmful) and the cellular site driving their switch, are still unclear. Here, we used a multidisciplinary approach to study the correlation between autophagy and apoptosis in renal NRK-52E cells exposed to cisPt. We showed two "sensitivity-thresholds" to cisPt, stating whether apoptosis or autophagy would develop: 10 μM dose of cisPt activated autophagy that preserved cell homeostasis; however 3-methyladenine co-administration affected cell viability and induced apoptosis. In contrast, 50 μM cisPt determined cell death by apoptosis, whereas the pre-conditioning with taurine contributed to cell rescue, delaying apoptosis and maintaining autophagy. Hence, autophagy protects NRK-52E cells from cisPt injury. By studying the expression of ER specific hallmarks, such as GRP78, GRP94 and GADD153/CHOP, we found a possible pivotal role of ER signaling modulation in the crosstalk between autophagy and apoptosis induced by cisPt. To the best of our knowledge, this is the first demonstration that taurine enhances autophagic protection against apoptosis by reducing ER stress, thus making it possible to develop new strategies to reduce severe cisPt-induced side-effects such as nephrotoxicity.     

A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10(-7) is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.     

Coexistence of multiple pathogen strains in stochastic epidemic models with density-dependent mortality

Stochastic differential equations that model an SIS epidemic with multiple pathogen strains are derived from a system of ordinary differential equations. The stochastic model assumes there is demographic variability. The dynamics of the deterministic model are summarized. Then the dynamics of the stochastic model are compared to the deterministic model. In the deterministic model, there can be either disease extinction, competitive exclusion, where only one strain persists, or coexistence, where more than one strain persists. In the stochastic model, all strains are eventually eliminated because the disease-free state is an absorbing state. However, if the population size and the initial number of infected individuals are sufficiently large, it may take a long time until all strains are eliminated. Numerical simulations of the stochastic model show that coexistence cases predicted by the deterministic model are an unlikely occurrence in the stochastic model even for short time periods. In the stochastic model, either disease extinction or competitive exclusion occur. The initial number of infected individuals, the basic reproduction numbers, and other epidemiological parameters are important determinants of the dominant strain in the stochastic epidemic model.     

Intracellular signaling is changed after clustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation

Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association.     

Correlated switch binding and signaling in bacterial chemotaxis

In Escherichia coli, swimming behavior is mediated by the phosphorylation state of the response regulator CheY. In its active, phosphorylated form, CheY exhibits enhanced binding to a switch component, FliM, at the flagellar motor, which induces a change from counterclockwise to clockwise flagellar rotation. When Ile(95) of CheY is replaced by a valine, increased clockwise rotation correlates with enhanced binding to FliM. A possible explanation for the hyperactivity of this mutant is that residue 95 affects the conformation of nearby residues that potentially interact with FliM. In order to assess this possibility directly, the crystal structure of CheY95IV was determined. We found that CheY95IV is structurally almost indistinguishable from wild-type CheY. Several other mutants with substitutions at position 95 were characterized to establish the structural requirements for switch binding and clockwise signaling at this position and to investigate a general relationship between the two properties. The various rotational phenotypes of these mutants can be explained solely by the amount of phosphorylated CheY bound to the switch, which was inferred from the phosphorylation properties of the mutant CheY proteins and their binding affinities to FliM. Combined genetic, biochemical, and crystallographic results suggest that residue 95 itself is critical in mediating the surface complementarity between CheY and FliM.     

Persistence of cooperatively stabilized signaling clusters drives T-cell activation

Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-gamma1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.     

Afferent signaling drives oxytocinergic preautonomic neurons and mediates training-induced plasticity

We showed previously that oxytocinergic (OTergic) projections from the hypothalamic paraventricular nucleus (PVN) to the dorsal brain stem mediate training-induced heart rate (HR) adjustments and that beneficial effects of training are blocked by sinoaortic denervation (SAD; Exp Physiol 94: 630-640; 1103-1113, 2009). We sought now to determine the combined effect of training and SAD on PVN OTergic neurons in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Rats underwent SAD or sham surgery and were trained (55% of maximal capacity) or kept sedentary for 3 mo. After hemodynamic measurements were taken at rest, rats were deeply anesthetized. Fresh brains were frozen and sliced to isolate the PVN; samples were processed for OT expression (real-time PCR) and fixed brains were processed for OT immunofluorescence. In sham rats, training improved treadmill performance and increased the gain of baroreflex control of HR. Training reduced resting HR (-8%) in both groups, with a fall in blood pressure (-10%) only in SHR rats. These changes were accompanied by marked increases in PVN OT mRNA expression (3.9- and 2.2-fold in WKY and SHR rats, respectively) and peptide density in PVN OTergic neurons (2.6-fold in both groups), with significant correlations between OT content and training-induced resting bradycardia. SAD abolished PVN OT mRNA expression and markedly reduced PVN OT density in WKY and SHR. Training had no effect on HR, PVN OT mRNA, or OT content following SAD. The chronic absence of inputs from baroreceptors and chemoreceptors uncovers the pivotal role of afferent signaling in driving both the plasticity and activity of PVN OTergic neurons, as well as the beneficial effects of training on cardiovascular control.     

Phase- and density-dependent population dynamics in Norwegian lemmings: interaction between deterministic and stochastic processes.

We analysed two 26-year long (1970-1995) time-series on annual population growth rates of Norwegian lemmings (Lemmus lemmus) from Finse, south Norway, using a threshold autoregressive (TAR) approach. We demonstrate that the population dynamics is both phase- and density-dependent. The phase-dependence accounts for the observed nonlinearity. We used the deduced stochastic model structure as a basis for evaluating the dynamic properties of this system. The dynamics is characterized either by limit cycles or chaos (the latter with a strong semi-periodic component). Stochasticity is seen to play an important role in the determination of the periodicity. The ecological implications of these statistical and mathematical results are discussed.     

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.     

Membrane clustering and the role of rebinding in biochemical signaling

In many cellular signaling pathways, key components form clusters at the cell membrane. Although much work has focused on the mechanisms behind such cluster formation, the implications for downstream signaling remain poorly understood. Here, motivated by recent experiments, we use particle-based simulation to study a covalent modification network in which the activating component is either clustered or randomly distributed on the membrane. We find that whereas clustering reduces the response of a single-modification network, it can enhance the response of a double-modification network. The reduction is a bulk effect: a cluster presents a smaller effective target to a substrate molecule in the bulk. The enhancement, on the other hand, is a local effect: a cluster promotes the rapid rebinding and second activation of singly activated substrate molecules. As such, the enhancement relies on frequent collisions on a short timescale, leading to an optimal ratio of diffusion to association that agrees with typical measured rates. We complement simulation with analytic results at both the mean-field and first-passage distribution levels. Our results emphasize the importance of spatially resolved models, showing that significant effects of spatial correlations persist even in spatially averaged quantities such as response curves.