An Experimental and Computational Study of the Effect of ActA Polarity on the Speed of Listeria monocytogenes Actin-based Motility

Listeria monocytogenes is a pathogenic bacterium that moves within infected cells and spreads directly between cells by harnessing the cell's dendritic actin machinery. This motility is dependent on expression of a single bacterial surface protein, ActA, a constitutively active Arp2,3 activator, and has been widely studied as a biochemical and biophysical model system for actin-based motility. Dendritic actin network dynamics are important for cell processes including eukaryotic cell motility, cytokinesis, and endocytosis. Here we experimentally altered the degree of ActA polarity on a population of bacteria and made use of an ActA-RFP fusion to determine the relationship between ActA distribution and speed of bacterial motion. We found a positive linear relationship for both ActA intensity and polarity with speed. We explored the underlying mechanisms of this dependence with two distinctly different quantitative models: a detailed agent-based model in which each actin filament and branched network is explicitly simulated, and a three-state continuum model that describes a simplified relationship between bacterial speed and barbed-end actin populations. In silico bacterial motility required a cooperative restraining mechanism to reconstitute our observed speed-polarity relationship, suggesting that kinetic friction between actin filaments and the bacterial surface, a restraining force previously neglected in motility models, is important in determining the effect of ActA polarity on bacterial motility. The continuum model was less restrictive, requiring only a filament number-dependent restraining mechanism to reproduce our experimental observations. However, seemingly rational assumptions in the continuum model, e.g. an average propulsive force per filament, were invalidated by further analysis with the agent-based model. We found that the average contribution to motility from side-interacting filaments was actually a function of the ActA distribution. This ActA-dependence would be difficult to intuit but emerges naturally from the nanoscale interactions in the agent-based representation

Control of Dendritic Morphogenesis by Trio in Drosophila melanogaster

Abl tyrosine kinase and its effectors among the Rho family of GTPases each act to control dendritic morphogenesis in Drosophila. It has not been established, however, which of the many GTPase regulators in the cell link these signaling molecules in the dendrite. In axons, the bifunctional guanine exchange factor, Trio, is an essential link between the Abl tyrosine kinase signaling pathway and Rho GTPases, particularly Rac, allowing these systems to act coordinately to control actin organization. In dendritic morphogenesis, however, Abl and Rac have contrary rather than reinforcing effects, raising the question of whether Trio is involved, and if so, whether it acts through Rac, Rho or both. We now find that Trio is expressed in sensory neurons of the Drosophila embryo and regulates their dendritic arborization. trio mutants display a reduction in dendritic branching and increase in average branch length, whereas over-expression of trio has the opposite effect. We further show that it is the Rac GEF domain of Trio, and not its Rho GEF domain that is primarily responsible for the dendritic function of Trio. Thus, Trio shapes the complexity of dendritic arbors and does so in a way that mimics the effects of its target, Rac

Exact Hybrid Particle/Population Simulation of Rule-Based Models of Biochemical Systems

Detailed modeling and simulation of biochemical systems is complicated by the problem of combinatorial complexity, an explosion in the number of species and reactions due to myriad protein-protein interactions and post-translational modifications. Rule-based modeling overcomes this problem by representing molecules as structured objects and encoding their interactions as pattern-based rules. This greatly simplifies the process of model specification, avoiding the tedious and error prone task of manually enumerating all species and reactions that can potentially exist in a system. From a simulation perspective, rule-based models can be expanded algorithmically into fully-enumerated reaction networks and simulated using a variety of network-based simulation methods, such as ordinary differential equations or Gillespie's algorithm, provided that the network is not exceedingly large. Alternatively, rule-based models can be simulated directly using particle-based kinetic Monte Carlo methods. This "network-free" approach produces exact stochastic trajectories with a computational cost that is independent of network size. However, memory and run time costs increase with the number of particles, limiting the size of system that can be feasibly simulated. Here, we present a hybrid particle/population simulation method that combines the best attributes of both the network-based and network-free approaches. The method takes as input a rule-based model and a user-specified subset of species to treat as population variables rather than as particles. The model is then transformed by a process of "partial network expansion" into a dynamically equivalent form that can be simulated using a population-adapted network-free simulator. The transformation method has been implemented within the open-source rule-based modeling platform BioNetGen, and resulting hybrid models can be simulated using the particle-based simulator NFsim. Performance tests show that significant memory savings can be achieved using the new approach and a monetary cost analysis provides a practical measure of its utility. © 2014 Hogg et al

Theoretical Model for Cellular Shapes Driven by Protrusive and Adhesive Forces

The forces that arise from the actin cytoskeleton play a crucial role in determining the cell shape. These include protrusive forces due to actin polymerization and adhesion to the external matrix. We present here a theoretical model for the cellular shapes resulting from the feedback between the membrane shape and the forces acting on the membrane, mediated by curvature-sensitive membrane complexes of a convex shape. In previous theoretical studies we have investigated the regimes of linear instability where spontaneous formation of cellular protrusions is initiated. Here we calculate the evolution of a two dimensional cell contour beyond the linear regime and determine the final steady-state shapes arising within the model. We find that shapes driven by adhesion or by actin polymerization (lamellipodia) have very different morphologies, as observed in cells. Furthermore, we find that as the strength of the protrusive forces diminish, the system approaches a stabilization of a periodic pattern of protrusions. This result can provide an explanation for a number of puzzling experimental observations regarding cellular shape dependence on the properties of the extra-cellular matrix

Cell motility: the integrating role of the plasma membrane

The plasma membrane is of central importance in the motility process. It defines the boundary separating the intracellular and extracellular environments, and mediates the interactions between a motile cell and its environment. Furthermore, the membrane serves as a dynamic platform for localization of various components which actively participate in all aspects of the motility process, including force generation, adhesion, signaling, and regulation. Membrane transport between internal membranes and the plasma membrane, and in particular polarized membrane transport, facilitates continuous reorganization of the plasma membrane and is thought to be involved in maintaining polarity and recycling of essential components in some motile cell types. Beyond its biochemical composition, the mechanical characteristics of the plasma membrane and, in particular, membrane tension are of central importance in cell motility; membrane tension affects the rates of all the processes which involve membrane deformation including edge extension, endocytosis, and exocytosis. Most importantly, the mechanical characteristics of the membrane and its biochemical composition are tightly intertwined; membrane tension and local curvature are largely determined by the biochemical composition of the membrane and the biochemical reactions taking place; at the same time, curvature and tension affect the localization of components and reaction rates. This review focuses on this dynamic interplay and the feedbacks between the biochemical and biophysical characteristics of the membrane and their effects on cell movement. New insight on these will be crucial for understanding the motility process

Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass

Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitative protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, B-1,3 glucanase, B-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed B-1,3(4) glucanase, B-1,3 glucanases, B-glucosidases, B-mannosidase, B-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo B-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. The participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.Peer reviewedMicrobiology and Molecular GeneticsBiochemistry and Microbiolog

Quantitative visualization of pectin distribution maps of peach fruits

Abstract Pectin content is an important quality index of fruits, as pectin content undergoes significant changes during the peach ripening process. The commonly used carbazole colorimetry method measures only the total content value of each kind of pectin for each pulp sample and cannot provide distribution maps of the pectin contents for the whole fruit pulp. This work used the hyperspectral imaging technique to quantitatively visualize the distribution maps of pectin contents inside peach pulp at the pixel level. The protopectin contents were well predicted, with the best residual predictive deviation of 2.264, whereas the predictions of the water-soluble pectin and the total pectin contents were not satisfied. On the basis of the best predictive model, the distribution maps of the protopectin contents were quantitatively visualized. A histogram of an example protopectin distribution revealed the existence of a wide range of protopectin contents in peach pulp. Our results show that hyperspectral imaging holds promise as a powerful alternative to the carbazole colorimetry method for measuring the spatial variations in the protopectin distribution inside peach pulp. The distribution maps could be used as a maturity indicator to understand and evaluate the ripening process of peach fruit in depth