Detailed simulations of cell biology with Smoldyn 2.1.

Most cellular processes depend on intracellular locations and random collisions of individual protein molecules. To model these processes, we developed algorithms to simulate the diffusion, membrane interactions, and reactions of individual molecules, and implemented these in the Smoldyn program. Compared to the popular MCell and ChemCell simulators, we found that Smoldyn was in many cases more accurate, more computationally efficient, and easier to use. Using Smoldyn, we modeled pheromone response system signaling among yeast cells of opposite mating type. This model showed that secreted Bar1 protease might help a cell identify the fittest mating partner by sharpening the pheromone concentration gradient. This model involved about 200,000 protein molecules, about 7000 cubic microns of volume, and about 75 minutes of simulated time; it took about 10 hours to run. Over the next several years, as faster computers become available, Smoldyn will allow researchers to model and explore systems the size of entire bacterial and smaller eukaryotic cells

Bridging Time Scales in Cellular Decision Making with a Stochastic Bistable Switch

Cellular transformations which involve a significant phenotypical change of the cell's state use bistable biochemical switches as underlying decision systems. In this work, we aim at linking cellular decisions taking place on a time scale of years to decades with the biochemical dynamics in signal transduction and gene regulation, occuring on a time scale of minutes to hours. We show that a stochastic bistable switch forms a viable biochemical mechanism to implement decision processes on long time scales. As a case study, the mechanism is applied to model the initiation of follicle growth in mammalian ovaries, where the physiological time scale of follicle pool depletion is on the order of the organism's lifespan. We construct a simple mathematical model for this process based on experimental evidence for the involved genetic mechanisms. Despite the underlying stochasticity, the proposed mechanism turns out to yield reliable behavior in large populations of cells subject to the considered decision process. Our model explains how the physiological time constant may emerge from the intrinsic stochasticity of the underlying gene regulatory network. Apart from ovarian follicles, the proposed mechanism may also be of relevance for other physiological systems where cells take binary decisions over a long time scale.Comment: 14 pages, 4 figure

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Bioengineering models of cell signaling

Strategies for rationally manipulating cell behavior in cell-based technologies and molecular therapeutics and understanding effects of environmental agents on physiological systems may be derived from a mechanistic understanding of underlying signaling mechanisms that regulate cell functions. Three crucial attributes of signal transduction necessitate modeling approaches for analyzing these systems: an ever-expanding plethora of signaling molecules and interactions, a highly interconnected biochemical scheme, and concurrent biophysical regulation. Because signal flow is tightly regulated with positive and negative feedbacks and is bidirectional with commands traveling both from outside-in and inside-out, dynamic models that couple biophysical and biochemical elements are required to consider information processing both during transient and steady-state conditions. Unique mathematical frameworks will be needed to obtain an integrated perspective on these complex systems, which operate over wide length and time scales. These may involve a two-level hierarchical approach wherein the overall signaling network is modeled in terms of effective "circuit" or "algorithm" modules, and then each module is correspondingly modeled with more detailed incorporation of its actual underlying biochemical/biophysical molecular interactions

Gated Diffusion-controlled Reactions

The binding and active sites of proteins are often dynamically occluded by motion of the nearby polypeptide. A variety of theoretical and computational methods have been developed to predict rates of ligand binding and reactivity in such cases. Two general approaches exist, "protein centric" approaches that explicitly treat only the protein target, and more detailed dynamical simulation approaches in which target and ligand are both treated explicitly. This mini-review describes recent work in this area and some of the biological implications

Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling