MTBindingSim: simulate protein binding to microtubules.

SUMMARY: Many protein-protein interactions are more complex than can be accounted for by 1:1 binding models. However, biochemists have few tools available to help them recognize and predict the behaviors of these more complicated systems, making it difficult to design experiments that distinguish between possible binding models. MTBindingSim provides researchers with an environment in which they can rapidly compare different models of binding for a given scenario. It is written specifically with microtubule polymers in mind, but many of its models apply equally well to any polymer or any protein-protein interaction. MTBindingSim can thus both help in training intuition about binding models and with experimental design. AVAILABILITY AND IMPLEMENTATION: MTBindingSim is implemented in MATLAB and runs either within MATLAB (on Windows, Mac or Linux) or as a binary without MATLAB (on Windows or Mac). The source code (licensed under the GNU General Public License) and binaries are freely available at http://mtbindingsim.googlecode.com. CONTACT: jphilip@nd.edu; cpence@nd.edu.

Nuclear mechanotransduction: response of the lamina to extracellular stress with implications in aging.

Mechnotransduction, the phenomenon by which cells respond to applied force, is necessary for normal cell processes and is implicated in the pathology of several diseases including atherosclerosis. The exact mechanisms which govern how forces can affect gene expression have not been determined, but putative direct force effects on the genome would require transduction through the nuclear lamina. In this study we show that nuclei in cells exposed to shear stress significantly change shape, upregulate nuclear lamins and move lamins from the nuclear interior to the nuclear periphery. We hypothesize that the augmentation of the nuclear lamina at the nuclear periphery protects the nuclear interior from the force and allows a nuclear adaptation to shear stress. We also investigate the shear stress response of nuclei in cells that have been transfected with lamin A Delta50, which significantly stiffens nuclei. Lamin A Delta50 causes the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS) and models many aspects of normal aging. We find that the presence of lamin A Delta50 in only 30% of cells greatly reduces the response of the nuclear lamina in all cells in the flow field. We suggest that cells expressing lamin A Delta50 lack the ability to adapt to flow and may prevent neighboring cells from adapting as well. These results provide insight into the development of cardiovascular disease both in patients with HGPS and in normal aging.

Using MTBindingSim as a tool for experimental planning and interpretation.

MTBindingSim is a program that enables users to simulate experiments in which proteins or other ligands (e.g., drugs) bind to microtubules or other polymers under various binding models. The purpose of MTBindingSim is to help researchers and students gain an intuitive understanding of binding behavior and design experiments to distinguish between different binding mechanisms. MTBindingSim is open-source, freely available software and can be found at bindingtutor.org/mtbindingsim. This chapter first describes the capabilities of MTBindingSim, including the experimental designs and protein-binding models that it simulates, and then discusses two examples in which MTBindingSim is utilized in an experimental context. In the first, MTBindingSim is used to investigate potential explanations for unusual behavior observed in the binding of the neuronal protein Tau to microtubules, demonstrating that some potential explanations are incompatible with the experimental data. In the second example, MTBindingSim is used to design experiments to examine the question of whether the plus-end tracking protein EB1 binds preferentially to the microtubule seam.

Biochemical evidence that human EB1 does not bind preferentially to the microtubule seam.

EB1 is a highly conserved microtubule (MT) plus end tracking protein (+TIP) involved in regulating MT dynamics, but the mechanisms of its effects on MT polymerization remain undefined. Resolving this question requires understanding how EB1 interacts with MTs. Previous electron microscopy of the S. pombe EB1 homolog Mal3p suggested that Mal3p binds specifically to the MT seam, implying that EB1 family members promote MT polymerization by stabilizing the seam. However, more recent electron microscopy indicates that Mal3p binds everywhere except the seam. Neither set of experiments investigated the behavior of human EB1, or provided an explanation for why these studies arrived at different answers. To resolve these questions, we have used a combination of MT-binding assays and theoretical modeling with MTBindingSim. Our results indicate that human EB1 binds to the lattice, consistent with the recent Mal3p results, and show that Mal3p-binding assays that were previously interpreted as evidence for preferential seam binding are equally consistent with weak lattice binding. In addition, we used analytical ultracentrifugation to investigate the possibility that the EB1 monomer-dimer equilibrium might contribute to EB1 binding behavior, and determined that the EB1 dimerization dissociation constant is approximately 90 nM. We and others find that the cellular concentration of EB1 is on the order of 200 nM, suggesting that a portion of EB1 may be monomeric at physiological concentrations. These observations lead us to suggest that regulation of EB1 dimerization might play a role in controlling EB1 function.