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.

Factor XA binding to phosphatidylserine-containing membranes produces an inactive membrane-bound dimer.

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.

Mechanical and biochemical modeling of cortical oscillations in spreading cells.

Actomyosin-based cortical contractility is a common feature of eukaryotic cells and is involved in cell motility, cell division, and apoptosis. In nonmuscle cells, oscillations in contractility are induced by microtubule depolymerization during cell spreading. We developed an ordinary differential equation model to describe this behavior. The computational model includes 36 parameters. The values for all but two of the model parameters were taken from experimental measurements found in the literature. Using these values, we demonstrate that the model generates oscillatory behavior consistent with current experimental observations. The rhythmic behavior occurs because of the antagonistic effects of calcium-induced contractility and stretch-activated calcium channels. The model makes several experimentally testable predictions: 1), buffering intracellular calcium increases the period and decreases the amplitude of cortical oscillations; 2), increasing the number or activity of stretch activated channels leads to an increase in period and amplitude of cortical oscillations; 3), inhibiting Ca(2+) pump activity increases the period and amplitude of oscillations; and 4), a threshold exists for the calcium concentration below which oscillations cease.

Visinets: a web-based pathway modeling and dynamic visualization tool.

In this report we describe a novel graphically oriented method for pathway modeling and a software package that allows for both modeling and visualization of biological networks in a user-friendly format. The Visinets mathematical approach is based on causal mapping (CMAP) that has been fully integrated with graphical interface. Such integration allows for fully graphical and interactive process of modeling, from building the network to simulation of the finished model. To test the performance of Visinets software we have applied it to: a) create executable EGFR-MAPK pathway model using an intuitive graphical way of modeling based on biological data, and b) translate existing ordinary differential equation (ODE) based insulin signaling model into CMAP formalism and compare the results. Our testing fully confirmed the potential of the CMAP method for broad application for pathway modeling and visualization and, additionally, showed significant advantage in computational efficiency. Furthermore, we showed that Visinets web-based graphical platform, along with standardized method of pathway analysis, may offer a novel and attractive alternative for dynamic simulation in real time for broader use in biomedical research. Since Visinets uses graphical elements with mathematical formulas hidden from the users, we believe that this tool may be particularly suited for those who are new to pathway modeling and without the in-depth modeling skills often required when using other software packages.

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.

Causal mapping as a tool to mechanistically interpret phenomena in cell motility: application to cortical oscillations in spreading cells.

Biological processes that occur at the cellular level and consist of large numbers of interacting elements are highly nonlinear and generally involve multiple time and spatial scales. The quantitative description of these complex systems is of great importance but presents large challenges. We outline a new systems biology approach, causal mapping (CMAP), which is a coarse-grained biological network tool that permits description of causal interactions between the elements of the network and overall system dynamics. On one hand, the CMAP is an intermediate between experiments and physical modeling, describing major requisite elements, their interactions and paths of causality propagation. On the other hand, the CMAP is an independent tool to explore the hierarchical organization of cell and the role of uncertainties in the system. It appears to be a promising easy-to-use technique for cell biologists to systematically probe verbally formulated qualitative hypotheses. We apply the CMAP to study the phenomenon of contractility oscillations in spreading cells in which microtubules have been depolymerized. The precise mechanism by which these oscillations are governed by a complex mechano-chemical system is not known but the data observed in experiments can be described by a CMAP. The CMAP suggests that the source of the oscillations results from the opposing effects of Rho activation leading to a decreased level of myosin light chain phosphatase and a cyclic calcium influx caused by increased membrane tension and leading to a periodically enhanced activation of myosin light chain kinase.

Analysis method for measuring submicroscopic distances with blinking quantum dots.

A method is described that takes advantage of the intermittency ("blinking") in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment.

Detecting microdomains in intact cell membranes.

Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

Cooperative roles of factor V(a) and phosphatidylserine-containing membranes as cofactors in prothrombin activation.

Activation of prothrombin, as catalyzed by the prothrombinase complex (factor X(a), enzyme; factor V(a) and phosphatidylserine (PS)-containing membranes, cofactors), involves production and subsequent proteolysis of two possible intermediates, meizothrombin (MzII(a)) and prethrombin 2 plus fragment 1.2 (Pre2 & F1.2). V(max), K(m), or V(max)/K(m) for all four proteolytic steps was determined as a function of membrane-phospholipid concentration. Proteolysis was monitored using a fluorescent thrombin inhibitor, a chromogenic substrate, and SDS-PAGE. The kinetic constants for the conversion of MzII(a) and Pre2 & F1.2 to thrombin were determined directly. Pre2 & F1.2 conversion was linear in substrate concentration up to 4 microm, whereas MzII(a) proteolysis was saturable. First order rate constants for formation of MzII(a) and Pre2 & F1.2 could not be determined directly and were determined from global fitting of the data to a parallel, sequential model, each step of which was treated by the Michaelis-Menten formalism. The rate of direct conversion to thrombin without release of intermediates from the membrane-V(a)-X(a) complex (i.e. "channeling") also was adjusted because both the membranes and factor V(a) have been shown to cause channeling. k(cat), K(m), or k(cat)/K(m) values were reported for one lipid concentration, for which all X(a) was likely incorporated into a X(a)-V(a) complex on a PS membrane. Comparing previous results, which were obtained either with factor V(a) (Boskovic, D. S., Bajzar, L. S., and Nesheim, M. E. (2001) J. Biol. Chem. 276, 28686-28693) or with membranes individually (Wu, J. R., Zhou, C., Majumder, R., Powers, D. D., Weinreb, G., and Lentz, B. R. (2002) Biochemistry 41, 935-949), with results presented here we conclude that both factor V(a) and PS-containing membranes induce similar rate increases and pathway changes. Moreover, we have determined: 1) factor V(a) has the greatest effect in enhancing rates of individual proteolytic events; 2) PS-containing membranes have the greatest role in increasing the preference for the MzII(a) versus Pre2 pathway; and 3) PS membranes cause approximately 50% of the substrate to be activated via channeling at 50 microm membrane concentration, but factor V(a) extends the range of efficient channeling to much lower or higher membrane concentrations.