Many aspects of the biochemistry and physiology of living cells have in the past been simulated by networks of reactions as though they were electronic circuits. In such studies, components such as receptors, enzymes, or metabolites are portrayed as being wired together in a spatially defined manner through enzymatic and other reactions. But it is clear that living circuitry is not like this; it has unique features such as a highly malleable internal architecture and the existence of a multitude of molecular states that differ in fundamental respects from those of silicon devices. Moreover, the wiring of the cell depends on the diffusive movement of myriad different molecules large and small through the watery interstices of the cytoplasm. In order to understand such systems, we need experimental techniques that can identify individual molecules and track their locations and movements within the cell. Moreover, once data of this kind are obtained we will need advanced computational methods by which spatial locations and diffusive movements of individual molecules can be represented.We recently developed a computer program for the study of intracellular reactions that allows us to take into account both the spatial location of proteins and protein complexes and their diffusive movements (1). This program uses an approach known as Brownian dynamics, in which molecules are treated as individuals rather than as concentrations and space is treated continuously instead of being subdivided into finite elements (10). Our program is called Smoldyn, for Smoluchowski dynamics, because it is based on a theory for the diffusive encounter of molecules in solution developed by the Polish physicist Marjan Smoluchowski (27). Since molecules are treated as individuals, this program can accurately capture stochastic behavior and also simulate diffusive gradients naturally and accurately. We were also inspired in our work by the MCell program, which has been developed to account for ionic and molecular events occurring within neuromuscular synapses (32). However, Smoldyn has certain advantages over MCell for our purposes since it allows reactions to occur between diffusing molecules in solution (in the current version of MCell, reactions occur only at membrane surfaces). Moreover, the code of Smoldyn, unlike that of MCell, is publicly available (http://sahara.lbl.gov/ϳsandrews/software.html).In this paper, we describe the application of Smoldyn to the well-characterized phenomenon of bacterial chemotaxis in Escherichia coli. We first predict the activity of the cluster of chemotactic receptors at one end of the cell in response to different stimulus conditions by using previously described software. We then use the temporal activity profile created in this way as an input to Smoldyn to calculate the locations of the diffusing molecules CheY, CheYp, and CheZ within the cell. As a first demonstration of the capabilities of this program, we report a series of simulations in which the diffusion of the signaling protein CheYp is followe...

Our results display the potential use of computer-based bacteria as experimental objects for exploring subtleties of chemotactic behavior.

How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.DOI: http://dx.doi.org/10.7554/eLife.00668.001

The mariner family is probably the most widely distributed family of transposons in nature. Although these transposons are related to the well-studied bacterial insertion elements, there is evidence for major differences in their reaction mechanisms. We report the identification and characterization of complexes that contain the Himar1 transposase bound to a single transposon end. Titrations and mixing experiments with the native transposase and transposase fusions suggested that they contain different numbers of transposase monomers. However, the DNA protection footprints of the two most abundant single-end complexes are identical. This indicates that some transposase monomers may be bound to the transposon end solely by protein-protein interactions. This would mean that the Himar1 transposase can dimerize independently of the second transposon end and that the architecture of the synaptic complex has more in common with V(D)J recombination than with bacterial insertion elements. Like V(D)J recombination and in contrast to the case for bacterial elements, Himar1 catalysis does not appear to depend on synapsis of the transposon ends, and the single-end complexes are active for nicking and probably for cleavage. We discuss the role of this single-end activity in generating the mutations that inactivate the vast majority of mariner elements in eukaryotes.The Tc1/mariner superfamily of transposons consists of the Tc1 and mariner families of elements in eukaryotes and the more distantly related IS630-like elements in bacteria (25, 39). Members of the superfamily have a single transposase gene expressed in the germ line and/or the soma, transpose via a "cut and paste" DNA intermediate, and duplicate a TA dinucleotide upon insertion. This is probably the most widespread family of transposons in nature: members have been identified in bacteria (IS630), ciliates, fungi, plants, and most animal phyla, from Porifera (sponges) to humans. Although mariner elements are widespread, they are unevenly distributed in closely related species and the vast majority are inactive because of mutations. This suggests that they have an unusual life style that involves a high rate of horizontal transfer to new hosts, followed by a burst of transposition and subsequent vertical inactivation (37, 43).Homology-dependent gene silencing serves to control the spread of transposons, retroviruses, and other repetitive DNA elements in many eukaryotes. It is mediated by at least three distinct mechanisms, specifically DNA methylation, histone deacetylation, and RNA interference. However, depending on the identity of the organism and the repetitive element in question, these mechanisms are not always completely effective. As noted by McClintock, the rate of transposition in a given germ line can change over time, with cycles of activation and silencing lasting several generations (cited in reference 35). Furthermore, although all three mechanisms of homologydependent gene silencing operate in plants and are active against many retrotransposons, som...

Intracellular protein concentration gradients are generally thought to be unsustainable at steady-state due to diffusion. Here we show how protein concentration gradients can theoretically be sustained indefinitely through a relatively simple mechanism that couples diffusion to a spatially segregated kinase-phosphatase system. Although it is appreciated that such systems can theoretically give rise to phosphostate gradients, it has been assumed that they do not give rise to gradients in the total protein concentration. Here we show that this assumption does not hold if the two forms of protein have different diffusion coefficients. If, for example, the phosphorylated state binds selectively to a second larger protein or protein complex then a steady state gradient in total protein concentration will be created. We illustrate the principle with an analytical solution to the diffusion-reaction problem and by stochastic individual-based simulations using the Smoldyn program. We argue that protein gradients created in this way need to be considered in experiments using fluorescent probes and could in principle encode spatial information in the cytoplasm.

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G؉C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.

In the chemotaxis pathway of the bacterium Escherichia coli, signals are carried from a cluster of receptors to the flagellar motors by the diffusion of the protein CheY-phosphate (CheYp) through the cytoplasm. A second protein, CheZ, which promotes dephosphorylation of CheYp, partially colocalizes with receptors in the plasma membrane. CheZ is normally dimeric in solution but has been suggested to associate into highly active oligomers in the presence of CheYp. A model is presented here and supported by Brownian dynamics simulations, which accounts for these and other experimental data: A minority component of the receptor cluster (dimers of CheAshort) nucleates CheZ oligomerization and CheZ molecules move from the cytoplasm to a bound state at the receptor cluster depending on the current level of cellular stimulation. The corresponding simulations suggest that dynamic CheZ localization will sharpen cellular responses to chemoeffectors, increase the range of detectable ligand concentrations, and make adaptation more precise and robust. The localization and activation of CheZ constitute a negative feedback loop that provides a second tier of adaptation to the system. Subtle adjustments of this kind are likely to be found in many other signaling pathways.

We studied the spatial distribution, mobility, and trafficking of plasma membrane Ca 2ϩ ATPase-2 (PMCA2), a protein enriched in the hair cell apical membrane and essential for hair cell function. Using immunofluorescence, we determined that PMCA2 is enriched in the stereocilia and present at a relatively low concentration in the kinocilium and in the remaining apical membrane. Using an antibody to the extracellular domain of PMCA2 as a probe, we observed that PMCA2 diffuses laterally from the stereocilia membrane and is internalized at the apical cell border maintaining an estimated half-life of residency in the stereocilia of ϳ5-7 h. A computer simulation of our data indicates that PMCA2 has an estimated global diffusion coefficient of 0.01-0.005 m 2 /s. Using a green fluorescent protein tag, we observed that PMCA2 is rapidly delivered to the apical cell border from where it diffuses to the entire stereocilia surface. Fluorescence recovery after photobleaching experiments show that ϳ60% of PMCA2 in the stereocilia exhibit high mobility with a diffusion coefficient of 0.1-0.2 m 2 /s, whereas the remaining pool represents a relatively immobile fraction. These results suggest that PMCA2 molecules maintain transient interactions with other components of the stereocilia, and the mobile pool of PMCA2 mediates the exchange between the stereocilia and the removal and delivery sites at the periphery of the apical cell surface. This rapid turnover of a major stereocilia membrane protein matches the previously described rapid turnover of proteins of the stereocilia actin core, further demonstrating that these organelles undergo rapid continuous renewal.