Topology and robustness in the Drosophila segment polarity network

A complex hierarchy of genetic interactions converts a single-celled Drosophila melanogaster egg into a multicellular embryo with 14 segments. Previously, von Dassow et al. reported that a mathematical model of the genetic interactions that defined the polarity of segments (the segment polarity network) was robust (von Dassow et al. 2000). As quantitative information about the system was unavailable, parameters were sampled randomly. A surprisingly large fraction of these parameter sets allowed the model to maintain and elaborate on the segment polarity pattern. This robustness is due to the positive feedback of gene products on their own expression, which induces individual cells in a model segment to adopt different stable expression states (bistability) corresponding to different cell types in the segment polarity pattern. A positive feedback loop will only yield multiple stable states when the parameters that describe it satisfy a particular inequality. By testing which random parameter sets satisfy these inequalities, I show that bistability is necessary to form the segment polarity pattern and serves as a strong predictor of which parameter sets will succeed in forming the pattern. Although the original model was robust to parameter variation, it could not reproduce the observed effects of cell division on the pattern of gene expression. I present a modified version that incorporates recent experimental evidence and does successfully mimic the consequences of cell division. The behavior of this modified model can also be understood in terms of bistability in positive feedback of gene expression. I discuss how this topological property of networks provides robust pattern formation and how large changes in parameters can change the specific pattern produced by a network.     

Cytokinetic astralogy

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.Cell division, or cytokinesis, is accomplished via constriction of an equatorially localized contractile ring composed of filamentous actin and myosin II (Rappaport, 1996). Accurate division plane specification is essential to properly partition the cytoplasm and permit each daughter cell to receive a single copy of the genome. To ensure this accuracy, microtubules of the mitotic spindle signal to the cell cortex upon anaphase onset and promote assembly of the contractile ring between the separating chromosomes. The precise mechanism by which microtubules position the contractile ring, however, remains elusive.Early models on the nature of the spindle-derived signal proposed that astral rays (later found to be microtubules) position the division plane by either locally promoting contractility at the cell equator or inhibiting contractility at the cell poles (Rappaport, 1996). Recent evidence, though, suggests that distinct microtubule populations within a single cell provide multiple signals to promote accurate division (Canman et al., 2003; Bringmann and Hyman, 2005; Chen et al., 2008; Foe and von Dassow, 2008; von Dassow, 2009).The anaphase mitotic spindle contains several subtypes of microtubules, each of which is likely to contribute to division plane specification. Although kinetochore microtubules drive chromosome segregation during anaphase, nonkinetochore microtubules extend and maintain close proximity with the assembling central spindle (Mastronarde et al., 1993). Central spindle microtubules are highly stable (Salmon et al., 1976) and organize into an antiparallel bundled array between the separating chromosomes (Mastronarde et al., 1993). Preventing central spindle assembly usually results in a complete failure in cytokinesis, and prevents division plane specification in many cell types (Glotzer, 2005). Astral microtubules, however, are highly dynamic and grow out circumferentially from the centrosomes toward the cell cortex. Increasing evidence suggests that the astral microtubule signal inhibits contractility (see below; Canman et al., 2000; Kurz et al., 2002; Lewellyn et al., 2009).Regardless of the mechanism of division plane specification via microtubules, nearly all current models depend on direct contact between microtubules of the mitotic spindle and the cell cortex. Most of these models were based on observations that at the time of division plane specification, astral microtubules contact the cell cortex in nearly all systems studied. Nonkinetochore and/or central spindle microtubules have also been proposed to deliver critical contractile signals to the cell equator (Murata-Hori and Wang, 2002; Canman et al., 2003; Somers and Saint, 2003; Verbrugghe and White, 2004; Lewellyn et al., 2009; Vale et al., 2009). Yet in many cell types (especially early embryos), central spindle microtubules are at some distance from the cell cortex during division plane specification. Despite this, signal delivery for both astral and central spindle microtubules was proposed to occur via direct transport along microtubules to the cell cortex. The study of von Dassow et al. in this issue, however, indicates that accurate division plane specification does not require any close microtubule/cortical contact and may occur via a diffusion-based mechanism (see also Salmon and Wolniak, 1990).By treating echinoderm and Xenopus embryos with controlled levels of trichostatin A (TSA), which destabilizes acetylated dynamic microtubules via inhibition of the tubulin deacetylase HDAC6 (Matsuyama et al., 2002), von Dassow et al. (2009) were able to preferentially prevent astral microtubule growth while leaving central spindle microtubules intact. TSA treatment did not block anaphase onset or central spindle assembly, but resulted in the complete disruption of all direct microtubule contact with the cell cortex. Nevertheless, TSA-treated cells were able to undergo cytokinesis successfully (Fig. 1 A). The lack of astral microtubules in TSA-treated cells was also recapitulated by double centrosome ablation, and again the cells were able to undergo cytokinesis (Fig. 1 B). In both experiments, cytokinesis occurred in a timely manner, but the contractile ring was broader than in control cells. Together, these data suggest that spindle microtubules are sufficient to provide a diffusible stimulatory signal capable of defining the cell division plane without any direct contact with the cell cortex (von Dassow et al., 2009).Open in a separate windowFigure 1.Testing models of division plane specification by targeting distinct microtubule populations. By selectively eliminating astral microtubules with either controlled TSA-treatment (A) or by double centrosome ablation (B), von Dassow et al. (2009) provide strong evidence that microtubule contact with the cell cortex is not essential for successful cytokinesis. When a single centrosome was ablated, the division plane was displaced away from the ablated aster (B); this suggests that astral microtubules provide an inhibitory signal. Further, anucleate cells would only complete cytokinesis if the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (C).The authors noticed that cytokinesis occurred selectively at a position with reduced microtubule density in control cells; therefore, they explored the role of astral microtubules in division plane positioning. By selectively ablating one centrosome just before anaphase onset, von Dassow et al. (2009) were also able to provide strong support for an inhibitory role of astral microtubules in division plane specification. When a single centrosome was ablated, the division plane was displaced away from the remaining astral microtubules and toward the ablated centrosome (Fig. 1 B). Further evidence for an inhibitory role of astral microtubules in cytokinesis came from close examination of the intracentrosomal distance in anucleate cells that were able to undergo cytokinesis relative to those that were not. Cells were only able to undergo cytokinesis when the intracentrosomal distance exceeded the distance from the centrosomes to the cell cortex (Fig. 1 C), which suggests that cytokinesis requires an aster-free zone. The authors propose a mechanism in these anucleate cells whereby global activation of contractility drives division plane specification refined by a zone of astral separation (von Dassow et al., 2009). However, one possibility is that a central spindle still forms in these anucleate cells and thus provides the same diffusion-based signal that promotes division in cells without asters. Indeed, antiparallel arrays of bundled microtubules that resemble the central spindle are known to form between asters without intervening chromosomes in other systems (Savoian et al., 1999).To summarize, the results described by von Dassow et al. (2009) support a model in which central spindle microtubules provide a diffusible stimulatory signal to promote the assembly of a broad contractile ring, which is then refined by astral microtubules into a tight contractile ring. It is tempting to speculate on the molecular nature of this diffusible signal and mechanism of the astral refinement during cytokinesis. Signaling via the small GTPase Rho is required for cytokinesis and is dependent on spindle microtubules (Bement et al., 2005; Piekny et al., 2005). von Dassow et al. (2009) showed that in TSA-treated cells lacking astral microtubules, the equatorial zone of active Rho GTPase is broader relative to control cells. Rho activation is promoted (at least in part) via the central spindle–localized GTP exchange factor, ECT2 (Glotzer, 2005). In parallel, the GTPase-activating protein (GAP) CYK4/MgcRacGAP also associates with the central spindle, where it acts to both limit the zone of Rho activity (Miller and Bement, 2009) and to promote the inactivation of another small GTPase, Rac (D''Avino et al., 2004; Yoshizaki et al., 2004; Canman et al., 2008). Perhaps in parallel to central spindle mediated activation of Rho signaling, local inactivation of the inhibitory Rac signal via CYK4 GAP activity would further specify the division plane, even at a distance (Fig. 2). When the dynamic asters are present, they could then additionally amplify Rac signaling at the cell poles via a similar mechanism to what occurs during cell motility (Wittmann and Waterman-Storer, 2001). This local feedback loop would reinforce the positive signal coming from the central spindle via Rho activation and could help delimit active Rho at the cell equator (Fig. 2). Certainly, understanding how Rho activation can be propagated to the cell cortex via diffusion in such an accurate manner will be a major future challenge.Open in a separate windowFigure 2.Model for central spindle–mediated signaling via Rho family small GTPases. Central spindle–localized guanine nucleotide exchange factor ECT2 leads to Rho activation at the cell equator. At the same time, central spindle–localized CYK4 (a Rho family GAP) would also locally inactivate the inhibitory Rac signal. Further refinement of the zone of active Rho by astral microtubule–activated Rac could then sharpen the Rho zone into a tight contractile ring.     

How do site-specific DNA-binding proteins find their targets?

Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore ‘searching’ through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the ‘facilitated diffusion’ model of Berg, Winter and von Hippel, showing how non-specific DNA–protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional ‘sliding’ motion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive ‘hopping’ motions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of ~100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.     

Design and constraints of the Drosophila segment polarity module: robust spatial patterning emerges from intertwined cell state switches

The Drosophila segment polarity genes constitute the last tier in the segmentation cascade; their job is to maintain the boundaries between parasegments and provide positional "read-outs" within each parasegment for the entire developmental history of the animal. These genes constitute a relatively well-defined network with a relatively well-understood patterning task. In a previous publication (von Dassow et al. 2000. Nature 406:188-192) we showed that a computer model predicts the segment polarity network to be a robust boundary-making device. Here we elaborate those findings. First, we explore the constraints among parameters that govern the network model. Second, we test architectural variants of the core network, and show that the network tolerates a wide variety of adjustments in design. Third, we evaluate several topologically identical models that incorporate more or less molecular detail, finding that more-complex models perform noticeably better than simplified ones. Fourth, we discuss two instances in which the failure of the network model to behave in a life-like fashion highlights mechanistic details that need further experimental investigation. We conclude with an explanation of how the segment polarity network can be understood as an interwoven conspiracy of simple dynamical elements, several bistable switches and a homeostat. The robustness with which the network as a whole maintains a spatial regime of stable cell state emerges from generic dynamical properties of these simple elements.     

Flow-induced structural transition in the beta-switch region of glycoprotein Ib

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue β-switch region of platelet glycoprotein Ibα, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a β-hairpin. Interaction between glycoprotein Ibα and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to β-hairpin transition in glycoprotein Ibα is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop→β-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.     

Aggressive interactions for a decentralized regulation of foraging activity in the social wasp Polistes versicolor

Physical aggression among nestmates is commonly observed in animal societies like Polistes paper wasps, where it can be used to suppress worker reproduction. There is no consensus about how individuals use aggression in contexts other than reproduction. In order to clarify this topic, the regulation of worker-foraging behavior was studied in the Neotropical eusocial wasp Polistes versicolor. By experimentally manipulating food demand, we found evidence that aggression is used as a decentralized mechanism of regulating foraging, because after food supplementation: (1) aggression levels and foraging rates decreased and (2) aggression received and exhibited by foragers and non-foragers decreased. P. versicolor can use aggression in both reproductive and foraging contexts. The conclusion is drawn that this species is a good model for understanding how individuals differ from aggression related to reproduction and foraging, which would allow understanding of the evolutionary shift in the function of aggression from being the mechanism of reproductive control to being co-opted for the decentralized, self-organized regulation of worker-foraging.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

Structural Origins of Misfolding Propensity in the Platelet Adhesive Von Willebrand Factor A1 Domain

The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3. We investigate the extent to which structural elements contributing to intermediate conformations can be identified using a residue-specific implementation of the structure-energy-equivalence-of-domains algorithm (SEED), which parses proteins of known structure into their constituent thermodynamically cooperative components using protein-group-specific, transfer free energies. The structural elements computed to contribute to the non-two-state character coincide with regions where Von Willebrand disease mutations induce misfolded molten globule conformations of the A1 domain. This suggests a mechanism for the regulation of rheological platelet adhesion to A1 based on cooperative flexibility of the α2 and α3 helices flanking the platelet GPIbα receptor binding interface.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision

No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus ΔH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus ΔH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus ΔH.     

Benefit of Shading by Nurse Plant Does Not Change along a Stress Gradient in a Coastal Dune

The proximity of adult neighbors often increases the performance of woody seedlings under harsh environmental conditions but this nurse plant effect becomes less intense when abiotic stress is alleviated, as predicted by the stress gradient hypothesis (SGH). Although some studies have tested how the net nurse effect is changed by stress, few studies have tested how the mechanism that drives the facilitative effect of nurse responds to changes in stress. We conducted field experiments in a subtropical coastal dune to test if shading drives the known nurse effect of adults of the tree Guapira opposita on seedling performance of another tree species, Ternstroemia brasiliensis. We transplanted T. brasiliensis seedlings to three neighbor environments: under a G. opposita crown, under artificial shade and without neighbor as a control. Furthermore, assuming that proximity to the seashore correlates with stress intensity, we tested if the potential shade-driven facilitation became less intense as stress decreased. Regardless of the proximity to the seashore, after a year, the survival of T. brasiliensis seedlings was twice as high when the seedlings were under G. opposita or under artificial shade compared to the control, indicating that the nurse effect is driven by shade and that this facilitation mechanism is constant along the stress gradient. However, G. opposita and artificial shade had a negative effect on seedlings growth. Overall, our results showed that the facilitation mechanism behind the nurse effect did not wane as the stress was reduced. Furthermore, in spite of the potential costs in terms of biomass production, our study highlights the potential of nurse plants and artificial shade as techniques to improve the survival of transplanted seedlings used in the restoration of degraded shrubland coastal dunes.     

Zebrafish as a model to understand autophagy and its role in neurological disease

In the past decade, the zebrafish (Danio rerio) has become a popular model system for the study of vertebrate development, since the embryos and larvae of this species are small, transparent and undergo rapid development ex utero, allowing in vivo analysis of embryogenesis and organogenesis. These characteristics can also be exploited by researchers interested in signaling pathways and disease processes and, accordingly, there is a growing literature on the use of zebrafish to model human disease. This model holds great potential for exploring how autophagy, an evolutionarily conserved mechanism for protein degradation, influences the pathogeneses of a range of different human diseases and for the evaluation of this pathway as a potential therapeutic strategy. Here we summarize what is known about the regulation of autophagy in eukaryotic cells and its role in neurodegenerative disease and highlight how research using zebrafish has helped further our understanding of these processes.     

Temperature-sensitive mechanism regulating diapause in Heliothis zea

Pupal diapause in Heliothis zea is regulated by a temperature-sensitive mechanism which prevents ecdysone production despite the release of prothoracicotropic hormone. To determine how this mechanism functioned, donor prothoracic glands were implanted into prothoracic gland-ablated hosts to test their ability to produce ecdysone in a diapause-sustaining temperature of 19°C. Results of these experiments ruled out the possibility that ecdysis production was regulated by the nervous system or by a mechanism intrinsic to the prothoracic glands, and suggested that a humoral factor was required for diapause termination.Haemolymph injection experiments supported this humoral factor hypothesis, i.e. haemolymph from non-diapausing donor pupae terminated diapause in hosts maintained at 19°C, whereas haemolymph from diapausing donor pupae had no such effect. These findings indicate that the temperature-sensitive mechanism regulating H. zea diapause functions by controlling the availability of a humoral factor necessary for ecdysone production by the prothoracic glands.     

Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. In this paper, we report a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. We show that the inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, we identify here a mechanism controlling the morphogenesis of a squamous epithelium.     

Innate immune sensing of nucleic acids from pathogens

The innate immune system is important as the first line of defense to sense invading pathogens. Nucleic acids represent critical pathogen signatures that trigger a host proinflammatory immune response. Much progress has been made in understanding how DNA and RNA trigger host defense countermeasures, however, several aspects of how cytosolic nucleic acids are sensed remain unclear. This special issue reviews how the host innate immune system senses nucleic acids from Brucella abortus, Mycobacterium sp and Legionella pneumophila, viral DNA, the role of STING in DNA sensing and inflammatory diseases and the mechanism of viral RNA recognition by the small interfering RNA pathway in Drosophila melanogaster.     

Flow-Induced β-Hairpin Folding of the Glycoprotein Ibα β-Switch

Flow-induced shear has been identified as a regulatory driving force in blood clotting. Shear induces β-hairpin folding of the glycoprotein Ibα β-switch which increases affinity for binding to the von Willebrand factor, a key step in blood clot formation and wound healing. Through 2.1-μs molecular dynamics simulations, we investigate the kinetics of flow-induced β-hairpin folding. Simulations sampling different flow velocities reveal that under flow, β-hairpin folding is initiated by hydrophobic collapse, followed by interstrand hydrogen-bond formation and turn formation. Adaptive biasing force simulations are employed to determine the free energy required for extending the unfolded β-switch from a loop to an elongated state. Lattice and freely jointed chain models illustrate how the folding rate depends on the entropic and enthalpic energy, the latter controlled by flow. The results reveal that the free energy landscape of the β-switch has two stable conformations imprinted on it, namely, loop and hairpin—with flow inducing a transition between the two.