Anthony J. C. Ladd - US grants

PROPOSAL NO.: 0505929
PRINCIPAL INVESTIGATOR: A. Ladd
INSTITUTION NAME: University of Florida

MULTI-SCALE MODELING OF CHEMICAL-TO-MECHANICAL ENERGY CONVERSION IN ACTIN-BASED MOTILITY

This grant is to develop and validate a biologically relevant, multi-scale model of force generation by polymerization of the biopolymer, actin. Monomeric actin polymerizes into stiff filaments from surface-bound components, which crosslink and propel the surface forward. How the chemical energy involved in monomer addition is converted into mechanical work is critical in understanding cell motility, as well as for exploiting actin-based motility for micro-/nanoscale sensors and actuators. The extension of the wormlike model of the actin filaments to incorporate bending and torsion, as well as the incorporation of the hydration and gel will be studied. Intellectual merits include: incorporation of molecular-level kinetics and energetics into a mesoscale model of polymerizing and cross-linking filaments; the design of a computational framework for modeling the mechanical properties of solutions of stiff biopolymers such as actin, accounting for its resistance to bending and torsion, position and orientation-dependent chemical functionalization along the molecular backbone; and the coupling of the polymer dynamics to the surrounding solvent. Time-dependent variations in concentration of critical components of the polymerization process will also be studied. The broader impacts of the work include establishment of new collaborations between biochemists, physicists, and bioengineers studying actin networks, and chemical engineers developing numerical methods to simulate polymer solutions. New understanding of the coupling between filament elongation and force generation will be valuable in designing technological applications, such as microscale sensors and actuators using linear molecular motors based on actin motility. A small symposium will be organized to promote an objective discussion of the merits of various computational approaches to polymer simulations. The research will contribute to the education and training of graduate students in a collaborative, multidisciplinary environment and undergraduate students will participate in making specific calculations for software development and applications.

Award 1067072
PIs: Butler

An innovative research program combining theory, simulations, and experiments is pursued to investigate the role of hydrodynamic interactions in the electrophoresis of polyelectrolytes such as DNA. In capillary
electrophoresis of DNA, the molecules are spherical on average and the hydrodynamic interactions among various segments of DNA are exponentially screened - the flow fields generated by the backbone charges and surrounding counterions cancel. However if the DNA is stretched, for example by a pressure driven flow applied in conjunction with the electric field, then a migration transverse to the flow and field lines is observed experimentally.

The commonly held assumption that electric fields do not generate any long-range flow in the fluid surrounding a charged molecule is contradicted by theoretical work dating back to Debye; there is in
fact a dipolar flow generated by the polarization of the charge density by the electric field. Although this flow is weak in comparison to the electrophoretic velocity, its orientational dependence provides a means for elongated polyelectrolytes to migrate perpendicular to the flow and field lines. Our research is driven by the hypothesis that this polarization flow is responsible for several phenomena that cannot be explained without a long-range, fluid-mediated interaction between distant segments of the polyelectrolyte. Some of these phenomena have not yet been observed experimentally, such as a length-dependent electrophoretic mobility of an elongated polyelectrolyte, but they are predicted by simulations. Testing the underlying hypothesis by confirming the existence of these effects in laboratory experiments is a primary goal of the project.

Intellectual Merit: The simplest model of the polarization flow predicts a dipolar hydrodynamic interaction between distant segments of a charged polymer in an electric field. Recent numerical simulations based on this model showed that one can semi-quantitatively account for data collected from DNA experiments over a limited range of salt concentration, electric field, and flow rate. In the course of this work several new phenomena were discovered that have not been observed experimentally. The ongoing research examines the validity of the underlying model and explores the potential of this mechanism for manipulating the distribution of polyelectrolytes in microchannels.

Broader Impacts:
Research: This work is enhancing our theoretical understanding of polyelectrolytes and altering prevailing views regarding hydrodynamic screening in polyelectrolyte dynamics. The research also impacts a wide range of technologies that require the ability to control and position charged biopolymers within microchannels by creating additional possibilities for manipulation of polyelectrolytes that may be advantageous for applications such as enhancing adsorption in ?DNA biochips?. Another potential application uses the variation in migration velocity with chain length as a means for separating DNA strands by length.

Education: The program of research is integrated with our educational activities, which focus on preparing students to work in a world that increasingly depends upon international collaborations to efficiently advance science and develop new technologies. Activities include increasing the participation of students in international collaborations and meetings. Students of all levels are encouraged to become involved in advanced research within our laboratories; we have been, and continue, to work with an existing program for high school students at the University. In all of these activities, we emphasize the participation of underrepresented groups by actively seeking their involvement.

1236616
PI: Ladd

Microtubules have a thermal persistence length of several millimeters but in living cells they are frequently observed to be bent on length scales of just a few microns, indicating the action of large athermal forces. Most experimental force measurements with microtubules have been performed in vitro and are not directly relevant to what happens in vivo. As a result, the microtubule force balance remains unclear. This project aims to develop and validate a biologically relevant model for the forces generated on microtubules by motor proteins. The project offers a new paradigm for investigating force generation in cells, by severing individual microtubules with a laser in living cells, and using numerical models of microtubule mechanics to interpret the measurements.

Most essential mammalian cell functions, including migration and cell division, involve force generation by semi-flexible biopolymers called microtubules. An understanding of how the forces generated by motor proteins affect the shapes and thus the function of microtubules in the cell has applications in a number of fields: cellular transport, cytoskeletal mechanics and cell biology, engineering applications in nanobiotechnology and the understanding of diseases such as cancer. The project promotes a collaborative partnership between multiple investigators, involving experiments, modeling and simulations. The numerical model will form the basis for a long-term development of simulations of the mechanics of the cytoskeleton. In addition, the project contributes to the education and training of graduate and undergraduate students in a diverse, multidisciplinary environment.

This project is co-funded by the Physics of Living Systems in Physics Division.

Because genetic information can lead to the development of effective therapies to improve human health, it is important to develop methods for the cost-effective sequencing of an entire genome. Most biological molecules, such as proteins and DNA, carry a charge and can be readily transported through fluids by application of an electric field, a phenomenon known as electrophoresis. Microfluidic technologies can essentially miniaturize a chemical factory to the scale of a few centimeters and are widely used for genome sequencing and mapping. However, the samples still must be prepared using a labor-intensive process, involving cycles of chemical treatment, washing and centrifugation. A microfluidic system that could prepare samples by separating the DNA from proteins and other cellular debris would be faster, cheaper, and less error prone than currently used techniques. This research project combines theoretical and experimental investigations into the challenges involved in using electric fields within microfluidic devices to trap and separate DNA from cell samples.

When a polymer is subjected to a shear flow, it stretches and rotates to align at an angle to the flow. If an electric field is then applied in the opposite direction to the fluid flow, a charged polymer will migrate perpendicular to the axis of the fields. This action creates a strongly inhomogeneous distribution of DNA within the cross section of a capillary tube, and it can be exploited to trap DNA within a microfluidic device by suitable tuning of the flow and electric fields. This migration is specific to long, flexible, and charged molecules, of which DNA is the only such class of molecules in living cells. Hence, DNA is trapped within the device, while other molecules, such as proteins, pass through. The proposal aims to develop a better understanding of DNA (or other polyelectrolyte) migration in combined flow and electric fields, by using experiments and numerical simulations to validate the hypothesis that polyelectrolyte migration is driven by electrically-induced flows. The research will determine the magnitude of the cross-stream migration under a wide variety of conditions to optimize the design of microfluidic devices for DNA extraction and concentration from samples of whole blood. If DNA can be successfully purified from the lysate, this will be an important step towards integrating sample preparation and analysis on a single microfluidic chip.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.