The Control of the Controller: Molecular Mechanisms for Robust Perfect Adaptation and Temperature Compensation

Organisms have the property to adapt to a changing environment and keep certain components within a cell regulated at the same level (homeostasis). “Perfect adaptation” describes an organism's response to an external stepwise perturbation by regulating some of its variables/components precisely to their original preperturbation values. Numerous examples of perfect adaptation/homeostasis have been found, as for example, in bacterial chemotaxis, photoreceptor responses, MAP kinase activities, or in metal-ion homeostasis. Two concepts have evolved to explain how perfect adaptation may be understood: In one approach (robust perfect adaptation), the adaptation is a network property, which is mostly, but not entirely, independent of rate constant values; in the other approach (nonrobust perfect adaptation), a fine-tuning of rate constant values is needed. Here we identify two classes of robust molecular homeostatic mechanisms, which compensate for environmental variations in a controlled variable's inflow or outflow fluxes, and allow for the presence of robust temperature compensation. These two classes of homeostatic mechanisms arise due to the fact that concentrations must have positive values. We show that the concept of integral control (or integral feedback), which leads to robust homeostasis, is associated with a control species that has to work under zero-order flux conditions and does not necessarily require the presence of a physico-chemical feedback structure. There are interesting links between the two identified classes of homeostatic mechanisms and molecular mechanisms found in mammalian iron and calcium homeostasis, indicating that homeostatic mechanisms may underlie similar molecular control structures.     

Analyzing steady states of dynamics of bio-molecules from the structure of regulatory networks

Regulatory relations between biological molecules constitute complex network systems and realize diverse biological functions through the dynamics of molecular activities. However, we currently have very little understanding of the relationship between the structure of a regulatory network and its dynamical properties. In this paper we introduce a new method, named “linkage logic” to analyze the dynamics of network systems. By this method, we can restrict possible steady states of a given complex network system from the knowledge of regulatory linkages alone. The regulatory linkage simply specifies the list of variables that affect the dynamics of each variable. We formalize two aspects of the linkage logic: the “Principle of Compatibility” determines the upper limit of the diversity of possible steady states of the dynamics realized by a given network; the “Principle of Dependency” determines the possible combinations of states of the system. By combining these two aspects, (i) for a given network, we can identify a cluster of nodes that gives an alternative representation of the steady states of the whole system, (ii) we can reduce a given complex network into a simpler one without loss of the ability to generate the diversity of steady states, (iii) we can examine the consistency between the structure of network and observed set of steady states, and (iv) sometimes we can predict unknown states or unknown regulations from an observed set of steady states alone. We illustrate the method by several applications to an experimentally determined regulatory network for biological functions.     

High altitude adaptation of the schizothoracine fishes (Cyprinidae) revealed by the mitochondrial genome analyses

The schizothoracine fishes, also known as “mountain carps” are widely distributed in the Qinghai-Tibetan Plateau and its peripheral regions. Although they provide a prime example of high altitude adaptation, the phylogenetic relationships and the divergence times among these carp lineages are still controversial. Moreover, the genetic basis for high altitude adaptation is also poorly understood. In this study, we determined the mitochondrial genomes from two species of the schizothoracine fishes, representing a “morphologically primitive” clade and “morphologically specialized” clade, respectively. The phylogenetic tree and the divergence times were estimated within the evolutionary framework of the entire order Cypriniformes. Our results indicate a polyphylyetic relationship of the schizothoracine fishes and suggest two independent migration events into the Qinghai-Tibetan Plateau: one by the “morphologically primitive” clade in the Late Miocene and another by the “morphologically specialized” clade in the Eocene. Rapid speciation events of each clade from the Late Miocene to the Pliocene correspond to the timing of the geologic acceleration of the Qinghai-Tibetan Plateau. Interestingly, we found evidence for positive selection acting on the protein coding genes in the mitochondrial genomes of the “morphologically specialized” clade, implying a possible genetic basis for high altitude adaptation in this derived lineage of cypriniform fishes.     

Functionality and metagraph disintegration in boolean networks

We study regulatory networks of N genes giving rise to a vector expression profile v(t) in which each gene is Boolean; either on or off at any time. We require a network to produce a particular time sequence v(t) for t∈1,…,T and parameterize the complexity of such a genetic function by its duration T. We establish a number of new results regarding how functional complexity constrains genetic regulatory networks and their evolution. We find that the number of networks which generate a function decreases approximately exponentially with its complexity T and show there is a corresponding weakening of the robustness of those networks to mutations. These results suggest a limit on the functional complexity T of typical networks that is polynomial in N. However, we are also able to prove the existence of a, presumably small, class of networks in which this scales exponentially with N. We demonstrate that an increase in functional complexity T drives what we describe as a metagraph disintegration effect, breaking up the space of networks previously connected by neutral mutations and contrast this with what is found with less restrictive definitions of functionality. Our findings show how functional complexity could be a factor in shaping the evolutionary landscape and how the evolutionary history of a species constrains its future functionality. Finally we extend our analysis to functions with more exotic topologies in expression space, including “stars” and “trees”. We quantify how the properties of networks that give rise to these functions differ from those that produce linear functional paths with the same overall duration T.     

From heterochromatin islands to the NAD World: A hierarchical view of aging through the functions of mammalian Sirt1 and systemic NAD biosynthesis

For the past couple of decades, aging science has been rapidly evolving, and powerful genetic tools have identified a variety of evolutionarily conserved regulators and signaling pathways for the control of aging and longevity in model organisms. Nonetheless, a big challenge still remains to construct a comprehensive concept that could integrate many distinct layers of biological events into a systemic, hierarchical view of aging. The “heterochromatin island” hypothesis was originally proposed 10 years ago to explain deterministic and stochastic aspects of cellular and organismal aging, which drove the author to the study of evolutionarily conserved Sir2 proteins. Since a surprising discovery of their NAD-dependent deacetylase activity, Sir2 proteins, now called “sirtuins,” have been emerging as a critical epigenetic regulator for aging. In this review, I will follow the process of conceptual development from the heterochromatin island hypothesis to a novel, comprehensive concept of a systemic regulatory network for mammalian aging, named “NAD World,” summarizing recent studies on the mammalian NAD-dependent deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis. This new concept of the NAD World provides critical insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of aging in mammals.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Precision and kinetics of adaptation in bacterial chemotaxis

The chemotaxis network of the bacterium Escherichia coli is perhaps the most studied model for adaptation of a signaling system to persistent stimuli. Although adaptation in this system is generally considered to be precise, there has been little effort to quantify this precision, or to understand how and when precision fails. Using a Förster resonance energy transfer-based reporter of signaling activity, we undertook a systematic study of adaptation kinetics and precision in E. coli cells expressing a single type of chemoreceptor (Tar). Quantifiable loss of precision of adaptation was observed at levels of the attractant MeAsp as low 10 μM, with pronounced differences in both kinetics and precision of adaptation between addition and removal of attractant. Quantitative modeling of the kinetic data suggests that loss of precise adaptation is due to a slowing of receptor methylation as available modification sites become scarce. Moreover, the observed kinetics of adaptation imply large cell-to-cell variation in adaptation rates—potentially providing genetically identical cells with the ability to “hedge their bets” by pursuing distinct chemotactic strategies.     

Mutations in Desmin's Carboxy-Terminal “Tail” Domain Severely Modify Filament and Network Mechanics

Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l_p. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (l_p = 1.0-2.0 μm) and the wild-type desmin (l_p = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Mate choice for non-additive genetic benefits: a resolution to the lek paradox

In promiscuous mating systems, females often show a consistent preference to mate with one or a few males, presumably to acquire heritable genetic benefits for their offspring. However, strong directional selection should deplete additive genetic variation in fitness and consequently any benefit to expressing the preference by females (referred to as the lek paradox). Here, we provide a novel resolution that examines non-additive genetic benefits, such as overdominance or inbreeding, as a source of genetic variation. Focusing on the inbreeding coefficient f and overdominance effects, we use dynamic models to show that (1) f can be inherited from sire to offspring, (2) populations with females that express a mating preferences for outbred males (low f) maintain higher genetic variation than populations with females that mate randomly, and (3) preference alleles for outbred males can invade populations even when the alleles are associated with a fecundity cost. We show that non-additive genetic variation due to overdominance can be converted to additive genetic variation and becomes “heritable” when the frequencies of alternative homozygous genotypes at fitness loci deviate from equality. Unlike previous models that assume an infinite population size, we now show that genetic drift in finite populations can lead to the necessary deviations in the frequencies of homozygous genotypes. We also show that the “heritability of f,” and hence the benefit to a mating preference for non-additive genetic benefits, is highest in small populations and populations in which a smaller number of loci contribute to fitness via overdominance. Our model contributes to the solution of the lek paradox.     

Can a late bloomer become an early bird? Tools for flowering time adjustment

The transition from the vegetative to reproductive stage followed by inflorescence is a critical step in plant life; therefore, studies of the genes that influence flowering time have always been of great interest to scientists. Flowering is a process controlled by many genes interacting mutually in a genetic network, and several hypothesis and models of flowering have been suggested so far. Plants in temperate climatic conditions must respond mainly to changes in the day length (photoperiod) and unfavourable winter temperatures. To avoid flowering before winter, some plants exploit a specific mechanism called vernalization. This review summarises current achievements in the study of genes controlling flowering in the dicot model species thale cress (Arabidopsis thaliana), as well as in monocot model species rice (Oryza sativa) and temperate cereals such as barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). The control of flowering in crops is an attractive target for modern plant breeding efforts aiming to prepare locally well-adapted cultivars. The recent progress in genomics revealed the importance of minor-effect genes (QTLs) and natural allelic variation of genes for fine-tuning flowering and better cultivar adaptation. We briefly describe the up-to-date technologies and approaches that scientists may employ and we also indicate how these modern biotechnological tools and “-omics” can expand our knowledge of flowering in agronomically important crops.     

Pitfalls in comparisons of genetic distances: a case study of the avian family Acrocephalidae

Genetic distances are increasingly being used for identification and species delimitation, especially since the introduction of “barcoding”. While for phylogenetic inferences great care is generally taken to choose the best-fit evolutionary model, this is usually neglected in calculating genetic distances. Moreover, distances obtained from others than best-fit models, different lengths of sequences, and even different loci are often freely compared. We examined the influence of different methods on calculating genetic distances using mitochondrial cytochrome b sequences for the passerine family Acrocephalidae.We found substantial differences between: (1) corrected distances based on the best-fit model (TrN + Γ) vs. uncorrected p-distances; (2) distances calculated based on different parts of the same gene; and (3) distances calculated using the methods of “complete deletion” vs. “pairwise deletion” for sequences that included uncertain nucleotides. All these methodological differences affected comparisons between species and potential taxonomical conclusions.We suggest that (1) different loci are incomparable. (2) Only perfectly homologous regions (same length, same part of locus) should be compared. (3) In the case of sequences with some uncertain nucleotides, only distances calculated by the method of “complete deletion” are fully comparable. (4) Only distances based on the optimal substitution model should be used. (5) Even within the same locus, corrected genetic distances are unique to the study in which they are calculated, as they are conditional on the particular dataset and model selected for that dataset.     

The Impact of Gene Expression Variation on the Robustness and Evolvability of a Developmental Gene Regulatory Network

Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role.