Positive-feedback loops in cell cycle progression

A positive-feedback loop is a simple motif that is ubiquitous to the modules and networks that comprise cellular signaling systems. Signaling behaviors that are synonymous with positive feedback include amplification and rapid switching, maintenance, and the coherence of outputs. Recent advances have been made towards understanding how positive-feedback loops function, as well as their mechanistic basis in controlling eukaryotic cell cycle progression. Some of these advances will be reviewed here, including: how cyclin controls passage through Start and maintains coherence of G1/S regulon expression in yeast; how Polo-like kinase 1 activation is driven by Bora and Aurora A, and its expression is stimulated by Forkhead Box M1 in mammalian cells; and how some of the various dynamic behaviors of spindle assembly and anaphase onset can be produced.     

Response to host plant odors and aggregation pheromone by larvae of the Colorado potato beetle on a servosphere

Using a servosphere (locomotion compensator), locomotory behavior of Colorado potato beetle Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) larvae was measured in detail in response to pulsed and non-pulsed odors of hostplant and conspecific pheromone. Second instars showed decreased straightness of movement, and all larvae showed decreased speed, in response to potato odor. Change in straightness by 2nd instars was also significantly affected by the interaction of pheromone and pulsing treatments. Fourth instars showed increased positive anemotaxis in response to the combined hostplant and pheromone odors. To our knowledge, this is the first demonstration of larval behavioral responses to adult pheromone in a holometabolous insect.     

Competing docking interactions can bring about bistability in the MAPK cascade

Mitogen-activated protein kinases are crucial regulators of various cell fate decisions including proliferation, differentiation, and apoptosis. Depending on the cellular context, the Raf-Mek-Erk mitogen-activated protein kinase cascade responds to extracellular stimuli in an all-or-none manner, most likely due to bistable behavior. Here, we describe a previously unrecognized positive-feedback mechanism that emerges from experimentally observed sequestration effects in the core Raf-Mek-Erk cascade. Unphosphorylated/monophosphorylated Erk sequesters Mek into Raf-inaccessible complexes upon weak stimulation, and thereby inhibits cascade activation. Mek, once phosphorylated by Raf, triggers Erk phosphorylation, which in turn induces dissociation of Raf-inaccessible Mek-Erk heterodimers, and thus further amplifies Mek phosphorylation. We show that this positive circuit can bring about bistability for parameter values measured experimentally in living cells. Previous studies revealed that bistability can also arise from enzyme depletion effects in the Erk double (de)phosphorylation cycle. We demonstrate that the feedback mechanism proposed in this article synergizes with such enzyme depletion effects to bring about a much larger bistable range than either mechanism alone. Our results show that stable docking interactions and competition effects, which are common in protein kinase cascades, can result in sequestration-based feedback, and thus can have profound effects on the qualitative behavior of signaling pathways.     

An agent-based model to investigate the roles of attractive and repellent pheromones in ant decision making during foraging

Pharaoh's ants organise their foraging system using three types of trail pheromone. All previous foraging models based on specific ant foraging systems have assumed that only a single attractive pheromone is used. Here we present an agent-based model based on trail choice at a trail bifurcation within the foraging trail network of a Pharaoh's ant colony which includes both attractive (positive) and repellent (negative) trail pheromones. Experiments have previously shown that Pharaoh's ants use both types of pheromone. We investigate how the repellent pheromone affects trail choice and foraging success in our simulated foraging system. We find that both the repellent and attractive pheromones have a role in trail choice, and that the repellent pheromone prevents random fluctuations which could otherwise lead to a positive feedback loop causing the colony to concentrate its foraging on the unrewarding trail. An emergent feature of the model is a high level of variability in the level of repellent pheromone on the unrewarding branch. This is caused by the repellent pheromone exerting negative feedback on its own deposition. We also investigate the dynamic situation where the location of the food is changed after foraging trails are established. We find that the repellent pheromone has a key role in enabling the colony to refocus the foraging effort to the new location. Our results show that having a repellent pheromone is adaptive, as it increases the robustness and flexibility of the colony's overall foraging response.     

Autocrine loops with positive feedback enable context-dependent cell signaling

We describe a mechanism for context-dependent cell signaling mediated by autocrine loops with positive feedback. We demonstrate that the composition of the extracellular medium can critically influence the intracellular signaling dynamics induced by extracellular stimuli. Specifically, in the epidermal growth factor receptor (EGFR) system, amplitude and duration of mitogen-activated protein kinase (MAPK) activation are modulated by the positive-feedback loop formed by the EGFR, the Ras-MAPK signaling pathway, and a ligand-releasing protease. The signaling response to a transient input is short-lived when most of the released ligand is lost to the cellular microenvironment by diffusion and/or interaction with an extracellular ligand-binding component. In contrast, the response is prolonged or persistent in a cell that is efficient in recapturing the endogenous ligand. To study functional capabilities of autocrine loops, we have developed a mathematical model that accounts for ligand release, transport, binding, and intracellular signaling. We find that context-dependent signaling arises as a result of dynamic interaction between the parts of an autocrine loop. Using the model, we can directly interpret experimental observations on context-dependent responses of autocrine cells to ionizing radiation. In human carcinoma cells, MAPK signaling patterns induced by a short pulse of ionizing radiation can be transient or sustained, depending on cell type and composition of the extracellular medium. On the basis of our model, we propose that autocrine loops in this, and potentially other, growth factor and cytokine systems may serve as modules for context-dependent cell signaling.     

Robust growth of human immunodeficiency virus type 1 (HIV-1)

The persistence of human immunodeficiency virus type-1 (HIV-1) has long been attributed to its high mutation rate and the capacity of its resulting heterogeneous virus populations to evade host immune responses and antiviral drugs. However, this view is incomplete because it does not explain how the virus persists in light of the adverse effects mutations in the viral genome and variations in host functions can potentially have on viral functions and growth. Here we show that the resilience of HIV-1 can be credited, at least in part, to a robust response to perturbations that emerges as an intrinsic property of its intracellular development. Specifically, robustness in HIV-1 arises through the coupling of two feedback loops: a Rev-mediated negative feedback and a Tat-mediated positive feedback. By employing a mechanistic kinetic model for its growth we found that HIV-1 buffers the effects of many potentially detrimental variations in essential viral and cellular functions, including the binding of Rev to mRNA; the level of rev mRNA in the pool of fully spliced mRNA; the splicing of mRNA; the Rev-mediated nuclear export of incompletely-spliced mRNAs; and the nuclear import of Tat and Rev. The virus did not, however, perform robustly to perturbations in all functions. Notably, HIV-1 tended to amplify rather than buffer adverse effects of variations in the interaction of Tat with viral mRNA. This result shows how targeting therapeutics against molecular components of the viral positive-feedback loop open new possibilities and potential in the effective treatment of HIV-1.     

S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination.

Wild-type S. cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone. However, some mutants that are supersensitive to pheromone lack this ability to discriminate. In this study, we provide evidence for a novel role of alpha pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway. Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination. Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.     

Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations

The cell-cycle oscillator includes an essential negative-feedback loop: Cdc2 activates the anaphase-promoting complex (APC), which leads to cyclin destruction and Cdc2 inactivation. Under some circumstances, a negative-feedback loop is sufficient to generate sustained oscillations. However, the Cdc2/APC system also includes positive-feedback loops, whose functional importance we now assess. We show that short-circuiting positive feedback makes the oscillations in Cdc2 activity faster, less temporally abrupt, and damped. This compromises the activation of cyclin destruction and interferes with mitotic exit and DNA replication. This work demonstrates a systems-level role for positive-feedback loops in the embryonic cell cycle and provides an example of how oscillations can emerge out of combinations of subcircuits whose individual behaviors are not oscillatory. This work also underscores the fundamental similarity of cell-cycle oscillations in embryos to repetitive action potentials in pacemaker neurons, with both systems relying on a combination of negative and positive-feedback loops.     

Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana

The construction of synthetic cell-cell communication networks can improve our quantitative understanding of naturally occurring signaling pathways and enhance our capabilities to engineer coordinated cellular behavior in cell populations. Towards accomplishing these goals in eukaryotes, we developed and analyzed two artificial cell-cell communication systems in yeast. We integrated Arabidopsis thaliana signal synthesis and receptor components with yeast endogenous protein phosphorylation elements and new response promoters. In the first system, engineered yeast 'sender' cells synthesize the plant hormone cytokinin, which diffuses into the environment and activates a hybrid exogenous/endogenous phosphorylation signaling pathway in nearby engineered yeast 'receiver' cells. For the second system, the sender network was integrated into the receivers under positive-feedback regulation, resulting in population density-dependent gene expression (that is, quorum sensing). The combined experimental work and mathematical modeling of the systems presented here can benefit various biotechnology applications for yeast and higher level eukaryotes, including fermentation processes, biomaterial fabrication and tissue engineering.     

Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway.

The Saccharomyces cerevisiae G protein alpha subunit Gpa1p is involved in the response of both MATa and MAT alpha cells to pheromone. We mutagenized the GPA1 C terminus to characterize the receptor-interacting domain and to investigate the specificity of the interactions with the a- and alpha-factor receptors. The results are discussed with respect to a structural model of the Gpa1p C terminus that was based on the crystal structure of bovine transducin. Some mutants showed phenotypes different than the pheromone response and mating defects expected for mutations that affect receptor interactions, and therefore the mutations may affect other aspects of Gpa1p function. Most of the mutations that resulted in pheromone response and mating defects had similar effects in MATa and MAT alpha cells, suggesting that they affect the interactions with both receptors. Overexpression of the pheromone receptors increased the mating of some of the mutants tested but not the wild-type strain, consistent with defects in mutant Gpa1p-receptor interactions. The regions identified by the mating-defective mutants correlated well with the regions of mammalian G(alpha) subunits implicated in receptor interactions. The strongest mating type-specific effects were seen for mutations to proline and a mutation of a glycine residue predicted to form a C-terminal beta turn. The analogous beta turn in mammalian G(alpha) subunits undergoes a conformational change upon receptor interaction. We propose that the conformation of this region of Gpa1p differs during the interactions with the a- and alpha-factor receptors and that these mating type-specific mutations preclude the orientation necessary for interaction with one of the two receptors.     

Mathematical modeling of calcium homeostasis in yeast cells

In this study, based on currently available experimental observations on protein level, we constructed a mathematical model to describe calcium homeostasis in normally growing yeast cells (Saccharomyces cerevisiae). Simulation results show that tightly controlled low cytosolic calcium ion level can be a natural result under the general mechanism of gene expression feedback control. The calmodulin (a sensor protein) behavior in our model cell agrees well with relevant observations in real cells. Moreover, our model can qualitatively reproduce the experimentally observed response curve of real yeast cell responding to step-like disturbance in extracellular calcium ion concentration. Further investigations show that the feedback control mechanism in our model is as robust as it is in real cells.     

Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.     

性信息素对斜纹夜蛾雄蛾嗅觉相关基因 abp,pbp 和 or 表达的影响

【目的】信息素是个体之间传递信息的重要分子,研究性信息素对斜纹夜蛾 Spodoptera litura 嗅觉相关基因表达的影响对于增加性信息素作用机理的认识及其应用有重要的意义。【方法】本研究通过实时定量PCR（qRT-PCR）技术探究在性信息素刺激处理条件下,斜纹夜蛾成虫嗅觉相关基因 abp, pbp 和 or 表达水平的变化;利用性信息素在田间诱捕斜纹夜蛾雄蛾,并通过自动计数器记录每小时诱虫量,从而间接显示其交配行为的节律性。【结果】斜纹夜蛾雄虫触角中嗅觉相关基因abp, pbp 和 or 的表达具有节律特性。经性信息素化合物(Z9, Z11-14:OAc+Z9, Z12-14:OAc)刺激处理后,abp, pbp 和 or 表达量也发生了显著的改变。通过记录田间性信息素诱捕器在一天中不同时间段内诱捕的雄蛾数量,发现诱捕到的斜纹夜蛾也具有节律特性。【结论】基因表达水平上的节律特性可能与雄虫交配活动的节律相关联,说明性信息素处理也在一定程度上改变了其节律及其对性信息素的神经反应。这一结果也首次从基因水平证明性信息素的刺激处理提高了周缘神经系统对性信息素反应的敏感性,有助于我们理解性信息素作用的分子机理,对迷向及性诱和测报应用具有指导意义。     

Emergent synchronous bursting of oxytocin neuronal network

When young suckle, they are rewarded intermittently with a let-down of milk that results from reflex secretion of the hormone oxytocin; without oxytocin, newly born young will die unless they are fostered. Oxytocin is made by magnocellular hypothalamic neurons, and is secreted from their nerve endings in the pituitary in response to action potentials (spikes) that are generated in the cell bodies and which are propagated down their axons to the nerve endings. Normally, oxytocin cells discharge asynchronously at 1–3 spikes/s, but during suckling, every 5 min or so, each discharges a brief, intense burst of spikes that release a pulse of oxytocin into the circulation. This reflex was the first, and is perhaps the best, example of a physiological role for peptide-mediated communication within the brain: it is coordinated by the release of oxytocin from the dendrites of oxytocin cells; it can be facilitated by injection of tiny amounts of oxytocin into the hypothalamus, and it can be blocked by injection of tiny amounts of oxytocin antagonist. Here we show how synchronized bursting can arise in a neuronal network model that incorporates basic observations of the physiology of oxytocin cells. In our model, bursting is an emergent behaviour of a complex system, involving both positive and negative feedbacks, between many sparsely connected cells. The oxytocin cells are regulated by independent afferent inputs, but they interact by local release of oxytocin and endocannabinoids. Oxytocin released from the dendrites of these cells has a positive-feedback effect, while endocannabinoids have an inhibitory effect by suppressing the afferent input to the cells.     

Potential of a quantal response as a mechanism for oscillatory behavior: implications for our concepts of hormonal control mechanisms

This article describes the potential of a quantal (i.e., all-or-none) response as a model for understanding the interactions between endocrine, paracrine and autocrine hormones. We review the general features of continuous and discontinuous (i.e., oscillating) quantal models including the role of a threshold. In addition, we also describe a few of the many different biochemical mechanisms which may give rise to quantal behavior. One of the more attractive schemes involves the coordinate regulation of opposing biochemical pathways resulting from phosphorylation of hormone receptors and/or rate-limiting enzymes. At least one hormone receptor (i.e., that for insulin) and many rate-limiting enzymes which control the flow of metabolites through a variety of metabolic pathways can be phosphorylated at multiple sites by one or more protein kinases. Phosphorylation may enhance or inhibit the activities of these proteins depending on which sites are modified. Furthermore, since phosphorylation of some sites on a protein may enhance the ability of phosphoprotein phosphatases to dephosphorylate other sites responsible for biological activity of the protein, phosphorylation also has the potential to produce a discontinuous quantal response. Quantal response mechanisms may alter our notions of endocrine regulation. When a quantal response mechanism is applied to a simple negative feedback model similar to that which was originally postulated to explain the interactions between gonadotropin and steroid hormonal levels, the model can account for the oscillations in hormone levels even when the input is constant. Conversely, when a graded mechanism is applied to the same negative feedback model, the model will almost certainly result in constant hormone levels. Further, the model illustrates that small changes in rate constants and thresholds of response, amplification of hormonal signals, and degradation of intermediate regulators can produce large shifts in the output of the system. These may account for the variability in hormonal levels observed in some endocrine systems. Finally, the high sensitivity of the quantal response mechanism accounts for the data which suggest that gonadotropins may play permissive rather than causal roles in regulation of gonadal function. Since increasing evidence suggests that all cells of a given type may not be equal in terms of hormonal responsiveness, measurements of response in single cells over short time periods will be needed before the role of a quantal response can be determined and endocrine regulation will be fully understood.     

Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth

The inappropriate expression of the a-factor pheromone receptor (Ste3p) in the MATa cell leads to a striking inhibition of the yeast pheromone response, the result of a functional interaction between Ste3p and some MATa-specific protein. The present work identifies this protein as Asg7p. Normally, expression of Ste3p and Asg7p is limited to distinct haploid mating types, Ste3p to MATalpha cells and Asg7p to MATa cells. Artificial coexpression of the two in the same cell, either a or alpha, leads to dramatic inhibition of the pheromone response. Ste3p-Asg7p coexpression also perturbs the membrane trafficking of Ste3p: Ste3p turnover is slowed, a result of an Asg7p-mediated retardation of the secretory delivery of the newly synthesized receptor to the plasma membrane. However, in the absence of ectopic Ste3p expression, the asg7Delta mutation is without consequence either for pheromone signaling or overall mating efficiency of a cells. Indeed, the sole phenotype that can be assigned to MATa asg7Delta cells is observed following zygotic fusion to its alpha mating partner. Though formed at wild-type efficiency, zygotes from these pairings are morphologically abnormal. The pattern of growth is deranged: emergence of the first mitotic bud is delayed, and, in its place, growth is apparently diverted into a novel structure superficially resembling the polarized mating projection characteristic of haploid cells responding to pheromone. Together these results suggest a mechanism in which, following the zygotic fusion event, Ste3p and Asg7p gain access to one another and together act to repress the pheromone response, promoting the transition of the new diploid cell to vegetative growth.     

The mating-specific G(alpha) protein of Saccharomyces cerevisiae downregulates the mating signal by a mechanism that is dependent on pheromone and independent of G(beta)(gamma) sequestration.

It has been inferred from compelling genetic evidence that the pheromone-responsive G(alpha) protein of Saccharomyces cerevisiae, Gpa1, directly inhibits the mating signal by binding to its own beta(gamma) subunit. Gpa1 has also been implicated in a distinct but as yet uncharacterized negative regulatory mechanism. We have used three mutant alleles of GPA1, each of which confers resistance to otherwise lethal doses of pheromone, to explore this possibility. Our results indicate that although the G322E allele of GPA1 completely blocks the pheromone response, the E364K allele promotes recovery from pheromone treatment rather than insensitivity to it. This observation suggests that Gpa1, like other G(alpha) proteins, interacts with an effector molecule and stimulates a positive signal--in this case, an adaptive signal. Moreover, the Gpa1-mediated adaptive signal is itself induced by pheromone, is delayed relative to the mating signal, and does not involve sequestration of G(beta)(gamma). The behavior of N388D, a mutant form of Gpa1 predicted to be activated, strongly supports these conclusions. Although N388D cannot sequester beta(gamma), as evidenced by two-hybrid analysis and its inability to complement a Gpa1 null allele under normal growth conditions, it can stimulate adaptation and rescue a gpa1(delta) strain when cells are exposed to pheromone. Considered as a whole, our data suggest that the pheromone-responsive heterotrimeric G protein of S. cerevisiae has a self-regulatory signaling function. Upon activation, the heterotrimer dissociates into its two subunits, one of which stimulates the pheromone response, while the other slowly induces a negative regulatory mechanism that ultimately shuts off the mating signal downstream of the receptor.