RhoA regulates calcium-independent periodic contractions of the cell cortex.

When microtubules are depolymerized in spreading cells, they experience morphological oscillations characterized by a period of about a minute, indicating that normal interactions between the microfilament and microtubule systems have been significantly altered. This experimental system provides a test bed for the development of both fine- and coarse-grained models of complex motile processes, but such models need to be adequately informed by experiment. Using criteria based on Fourier transform analysis, we detect spontaneous oscillations in spreading cells. However, their amplitude and tendency to operate at a single frequency are greatly enhanced by microtubule depolymerization. Knockdown of RhoA and addition of various inhibitors of the downstream effector of RhoA, Rho kinase, block oscillatory behavior. Inhibiting calcium fluxes from endoplasmic reticulum stores and from the extracellular medium does not significantly affect the ability of cells to oscillate, indicating that calcium plays a subordinate regulatory role compared to Rho. We characterized the dynamic structure of the oscillating cell by light, fluorescence, and electron microscopy, showing how oscillating cells are dynamically polarized in terms of their overall morphology, f-actin and phosphorylated myosin light chain distribution, and nuclear position and shape. Not only will these studies guide future experiments, they will also provide a framework for the development of refined mathematical models of the oscillatory process.

In silico generation of alternative hypotheses using causal mapping (CMAP).

Previously, we introduced causal mapping (CMAP) as an easy to use systems biology tool for studying the behavior of biological processes that occur at the cellular and molecular level. CMAP is a coarse-grained graphical modeling approach in which the system of interest is modeled as an interaction map between functional elements of the system, in a manner similar to portrayals of signaling pathways commonly used by molecular cell biologists. CMAP describes details of the interactions while maintaining the simplicity of other qualitative methods (e.g., Boolean networks).In this paper, we use the CMAP methodology as a tool for generating hypotheses about the mechanisms that regulate molecular and cellular systems. Furthermore, our approach allows competing hypotheses to be ranked according to a fitness index and suggests experimental tests to distinguish competing high fitness hypotheses. To motivate the CMAP as a hypotheses generating tool and demonstrate the methodology, we first apply this protocol to a simple test-case of a three-element signaling module. Our methods are next applied to the more complex phenomenon of cortical oscillations observed in spreading cells. This analysis produces two high fitness hypotheses for the mechanism that underlies this dynamic behavior and suggests experiments to distinguish the hypotheses. The method can be widely applied to other cellular systems to generate and compare alternative hypotheses based on experimentally observed data and using computer simulations.