Singularity in polarization: rewiring yeast cells to make two buds.

For budding yeast to ensure formation of only one bud, cells must polarize toward one, and only one, site. Polarity establishment involves the Rho family GTPase Cdc42, which concentrates at polarization sites via a positive feedback loop. To assess whether singularity is linked to the specific Cdc42 feedback loop, we disabled the yeast cell's endogenous amplification mechanism and synthetically rewired the cells to employ a different positive feedback loop. Rewired cells violated singularity, occasionally making two buds. Even cells that made only one bud sometimes initiated two clusters of Cdc42, but then one cluster became dominant. Mathematical modeling indicated that, given sufficient time, competition between clusters would promote singularity. In rewired cells, competition occurred slowly and sometimes failed to develop a single "winning" cluster before budding. Slowing competition in normal cells also allowed occasional formation of two buds, suggesting that singularity is enforced by rapid competition between Cdc42 clusters.

Tipping the Balance: A Criticality Perspective.

Cell populations are often characterised by phenotypic heterogeneity in the form of two distinct subpopulations. We consider a model of tumour cells consisting of two subpopulations: non-cancer promoting (NCP) and cancer-promoting (CP). Under steady state conditions, the model has similarities with a well-known model of population genetics which exhibits a purely noise-induced transition from unimodality to bimodality at a critical value of the noise intensity σ2. The noise is associated with the parameter λ representing the system-environment coupling. In the case of the tumour model, λ has a natural interpretation in terms of the tissue microenvironment which has considerable influence on the phenotypic composition of the tumour. Oncogenic transformations give rise to considerable fluctuations in the parameter. We compute the λ-σ2 phase diagram in a stochastic setting, drawing analogies between bifurcations and phase transitions. In the region of bimodality, a transition from a state of balance to a state of dominance, in terms of the competing subpopulations, occurs at λ = 0. Away from this point, the NCP (CP) subpopulation becomes dominant as λ changes towards positive (negative) values. The variance of the steady state probability density function as well as two entropic measures provide characteristic signatures at the transition point.

Non-genetic heterogeneity, criticality and cell differentiation.

The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

Gene Silencing via RNA Interference in Cryptococcus.

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.

Early signatures of regime shifts in gene expression dynamics.

Recently, a large number of studies have been carried out on the early signatures of sudden regime shifts in systems as diverse as ecosystems, financial markets, population biology and complex diseases. The signatures of regime shifts in gene expression dynamics are less systematically investigated. In this paper, we consider sudden regime shifts in the gene expression dynamics described by a fold-bifurcation model involving bistability and hysteresis. We consider two alternative models, models 1 and 2, of competence development in the bacterial population B. subtilis and determine some early signatures of the regime shifts between competence and noncompetence. We use both deterministic and stochastic formalisms for the purpose of our study. The early signatures studied include the critical slowing down as a transition point is approached, rising variance and the lag-1 autocorrelation function, skewness and a ratio of two mean first passage times. Some of the signatures could provide the experimental basis for distinguishing between bistability and excitability as the correct mechanism for the development of competence.

Noise-induced regime shifts: A quantitative characterization.

Diverse complex dynamical systems are known to exhibit abrupt regime shifts at bifurcation points of the saddle-node type. The dynamics of most of these systems, however, have a stochastic component resulting in noise-driven regime shifts even if the system is away from the bifurcation points. In this paper, we propose a new quantitative measure, namely, the propensity transition point as an indicator of stochastic regime shifts. The concepts and the methodology are illustrated for the one-variable May model, a well-known model in ecology and the genetic toggle, a two-variable model of a simple genetic circuit. The general applicability and usefulness of the method for the analysis of regime shifts is further demonstrated in the case of the mycobacterial switch to persistence for which experimental data are available.

Molecular Pathogenesis of Nipah Virus.

Viral diseases are causing mayhem throughout the world. One of the zoonotic viruses that have emerged as a potent threat to community health in the past few decades is Nipah virus. Nipah viral sickness is a zoonotic disease whose main carrier is bat. This disease is caused by Nipah virus (NiV). It belongs to the henipavirous group and of the family paramyxoviridae. Predominantly Pteropus spp. is the carrier of this virus. It was first reported from the Kampung Sungai Nipah town of Malaysia in 1998. Human-to-human transmission can also occur. Several repeated outbreaks were reported from South and Southeast Asia in the recent past. In humans, the disease is responsible for rapid development of acute illness, which can result in severe respiratory illness and serious encephalitis. Therefore, this calls for an urgent need for health authorities to conduct clinical trials to establish possible treatment regimens to prevent any further outbreaks.

Emergent phenomena in living systems:A statistical mechanical perspective.

A natural phenomenon occurring in a living system is an outcome of the dynamics of the specific biological network underlying the phenomenon. The collective dynamics have both deterministic and stochastic components. The stochastic nature of the key processes like gene expression and cell differentiation give rise to fluctuations (noise) at the levels of the biomolecules, and this combined with nonlinear interactions gives rise to a number of emergent phenomena. In this review, we describe and discuss some of these phenomena which have the character of phase transitions in physical systems. We specifically focus on noise-induced transitions in a stochastic model of gene expression and in a population genetics model which have no analogs when the dynamics are solely deterministic in nature. Some of these transitions exhibit critical-point phenomena belonging to the mean-field Ising universality class of equilibrium phase transitions. A number of other examples, ranging from biofilms to homeostasis in adult tissues, are also discussed, which exhibit behaviour similar to critical phenomena in equilibrium and nonequilbrium phase transitions. The examples illustrate how the subject of statistical mechanics provides a bridge between theoretical models and experimental observations.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

Transient pulse formation in jasmonate signaling pathway.

The jasmonate (JA) signaling pathway in plants is activated as defense response to a number of stresses like attacks by pests or pathogens and wounding by animals. Some recent experiments provide significant new knowledge on the molecular detail and connectivity of the pathway. The pathway has two major components in the form of feedback loops, one negative and the other positive. We construct a minimal mathematical model, incorporating the feedback loops, to study the dynamics of the JA signaling pathway. The model exhibits transient gene expression activity in the form of JA pulses in agreement with experimental observations. The dependence of the pulse amplitude, duration and peak time on the key parameters of the model is determined computationally. The deterministic and stochastic aspects of the pathway dynamics are investigated using both the full mathematical model and a reduced version of it. We also compare the mechanism of pulse formation with the known mechanisms of pulse generation in some bacterial and viral systems.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p.

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

Osmolytes ameliorate the effects of stress in the absence of the heat shock protein Hsp104 in Saccharomyces cerevisiae.

Aggregation of the prion protein has strong implications in the human prion disease. Sup35p is a yeast prion, and has been used as a model protein to study the disease mechanism. We have studied the pattern of Sup35p aggregation inside live yeast cells under stress, by using confocal microscopy, fluorescence activated cell sorting and western blotting. Heat shock proteins are a family of proteins that are produced by yeast cells in response to exposure to stressful conditions. Many of the proteins behave as chaperones to combat stress-induced protein misfolding and aggregation. In spite of this, yeast also produce small molecules called osmolytes during stress. In our work, we tried to find the reason as to why yeast produce osmolytes and showed that the osmolytes are paramount to ameliorate the long-term effects of lethal stress in Saccharomyces cerevisiae, either in the presence or absence of Hsp104p.

Functional characteristics of a double positive feedback loop coupled with autorepression.

We study the functional characteristics of a two-gene motif consisting of a double positive feedback loop and an autoregulatory negative feedback loop. The motif appears in the gene regulatory network controlling the functional activity of pancreatic beta-cells. The model exhibits bistability and hysteresis in appropriate parameter regions. The two stable steady states correspond to low (OFF state) and high (ON state) protein levels, respectively. Using a deterministic approach, we show that the region of bistability increases in extent when the copy number of one of the genes is reduced from 2 to 1. The negative feedback loop has the effect of reducing the size of the bistable region. Loss of a gene copy, brought about by mutations, hampers the normal functioning of the beta-cells giving rise to the genetic disorder, maturity-onset diabetes of the young (MODY). The diabetic phenotype makes its appearance when a sizable fraction of the beta-cells is in the OFF state. Using stochastic simulation techniques we show that, on reduction of the gene copy number, there is a transition from the monostable ON to the ON state in the bistable region of the parameter space. Fluctuations in the protein levels, arising due to the stochastic nature of gene expression, can give rise to transitions between the ON and OFF states. We show that as the strength of autorepression increases, the ON --> OFF state transitions become less probable whereas the reverse transitions are more probable. The implications of the results in the context of the occurrence of MODY are pointed out.

Positive feedback, stochasticity and genetic competence.

A single gene, regulating its own expression via a positive feedback loop, constitutes a common motif in gene regulatory networks and signalling cascades. Recent experiments on the development of competence in the bacterial population B. subtilis show that the autoregulatory genetic module by itself can give rise to two types of cellular states. The states correspond to the low and high expression states of the master regulator ComK. The high expression state is attained when the ComK protein level exceeds a threshold value leading to a full activation of the autostimulatory loop. Stochasticity in gene expression drives the transitions between the two stable states. In this paper, we explain the appearance of bimodal protein distributions in B. subtilis cell population in the framework of three possible scenarios. In two of the cases, bistability provides the basis for binary gene expression. In the third case, the system is monostable in a deterministic description and stochasticity in gene expression is solely responsible for the appearance of the two expression states.

The p53-MDM2 network: from oscillations to apoptosis.

The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.

Criticality in cell differentiation.

Cell differentiation is an important process in living organisms. Differentiation is mostly based on binary decisions with the progenitor cells choosing between two specific lineages. The differentiation dynamics have both deterministic and stochastic components. Several theoretical studies suggest that cell differentiation is a bifurcation phenomenon, well-known in dynamical systems theory. The bifurcation point has the character of a critical point with the system dynamics exhibiting specific features in its vicinity. These include the critical slowing down, rising variance and lag-1 autocorrelation function, strong correlations between the fluctuations of key variables and non-Gaussianity in the distribution of fluctuations. Recent experimental studies provide considerable support to the idea of criticality in cell differentiation and in other biological processes like the development of the fruit fly embryo. In this review, an elementary introduction is given to the concept of criticality in cell differentiation. The correspondence between the signatures of criticality and experimental observations on blood cell differentiation in mice is further highlighted.

Stochastic model of transcription factor-regulated gene expression.

We consider a stochastic model of transcription factor (TF)-regulated gene expression. The model describes two genes, gene A and gene B, which synthesize the TFs and the target gene proteins, respectively. We show through analytic calculations that the TF fluctuations have a significant effect on the distribution of the target gene protein levels when the mean TF level falls in the highest sensitive region of the dose-response curve. We further study the effect of reducing the copy number of gene A from two to one. The enhanced TF fluctuations yield results different from those in the deterministic case. The probability that the target gene protein level exceeds a threshold value is calculated with the knowledge of the probability density functions associated with the TF and target gene protein levels. Numerical simulation results for a more detailed stochastic model are shown to be in agreement with those obtained through analytic calculations. The relevance of these results in the context of the genetic disorder haploinsufficiency is pointed out. Some experimental observations on the haploinsufficiency of the tumour suppressor gene, Nkx 3.1, are explained with the help of the stochastic model of TF-regulated gene expression.

Gene copy number and cell cycle arrest.

The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

Noise characteristics of feed forward loops.

A prominent feature of gene transcription regulatory networks is the presence in large numbers of motifs, i.e., patterns of interconnection, in the networks. One such motif is the feed forward loop (FFL) consisting of three genes X, Y and Z. The protein product x of X controls the synthesis of protein product y of Y. Proteins x and y jointly regulate the synthesis of z proteins from the gene Z. The FFLs, depending on the nature of the regulating interactions, can be of eight different types which can again be classified into two categories: coherent and incoherent. In this paper, we study the noise characteristics of FFLs using the Langevin formalism and the Monte Carlo simulation technique based on the Gillespie algorithm. We calculate the variances around the mean protein levels in the steady states of the FFLs and find that, in the case of coherent FFLs, the most abundant FFL, namely, the type-1 coherent FFL, is the least noisy. This is shown to be true for all parameter values when the FFLs operate above their thresholds of activation/repression. In the case of incoherent FFLs, no such general conclusion can be shown. The results suggest possible relationships between noise, functionality and abundance.