Ralph Nossal, PhD, Head, Section on Cell Biophysics
Dan Sackett, PhD, Staff Scientist
Hacène Boukari, PhD, Senior Fellow
Jennifer Galanis, MD, Postdoctoral Fellow
Matt Ferguson, MS, Predoctoral Fellow
Ariel Michelman-Ribeiro, MS, Predoctoral Fellow
Ron Skupsky, BA, Guest Researcher

We are studying elements of cell processes involved in signal transduction, protein trafficking, and cell motility. We are particularly interested in the way cellular activities are coordinated in space and time, and we develop and apply novel methodologies based on mathematical and physical principles in order to develop a better understanding of such phenomena. We focus on understanding the origination and transformations of supramolecular cellular assemblages such as protein-coated endocytic vesicles, metabolic signaling complexes, and cytoskeletal structures. We have thus constructed specialized fluorescence-based optical instrumentation to study dynamic supramolecular processes and have used advanced electromagnetic scattering techniques to examine structures on nanoscopic length scales. We also have developed mathematical models of specific aspects of cellular behavior.

Correlation spectroscopy and Fourier imaging (scattering)

Boukari, Nossal, Michelman-Ribeiro, Sackett; in collaboration with Horkay, Krueger, Lafer, Prasad, Schuck

We continue to develop fluorescence correlation spectroscopy (FCS) and similar methods to be used as tools for examining the properties of supramolecular biological assemblies. Recently, we have been studying the movement of probe molecules within hydrogels and other complex polymer networks. For example, we used FCS to obtain quantitative measures of the diffusion of various molecular sizes within poly(vinyl alcohol) (PVA) samples (solutions and gels) prepared at different polymer concentrations and cross-link densities. The measurements indicate that the diffusion rate is affected not only by the polymer concentration but also by the cross-link density of the gel. The results obtain even for certain very small probes, where we find that, for polymer concentrations below the gelation threshold, the diffusion rates remain unchanged with the addition of cross-linkers. However, above the threshold, movement of the probe particles through the gels is slower than in the corresponding polymer solutions. We are investigating how transient hydrogen bonding between the probes and the polymer chains might influence these results. We have also been using FCS and dynamic light scattering (DLS) to measure the hydrodynamic diameters of nanoscopic biological structures. We have been able to monitor the stability of tubulin rings that form in the presence of certain small antimitotic peptides, and, by combining FCS and analytical ultracentrifugation of the rings, we confirmed theories and computational code that provide values of hydrodynamic coefficients of particles of complex geometry. We subsequently used insights gained from these investigations to guide studies of the shapes of clathrin triskelions in solutions of different pH and salt concentrations. We showed that free triskelions have puckered shapes similar to, but notably different from, those of triskelions in reconstituted clathrin baskets. We recently verified our results with small angle neutron scattering (SANS), which, along with new methods of analysis, provides information about the rigidity of the triskelions as well as insight into their average shapes.

Membrane transformations underlying cell function

Nossal, Ferguson, Boukari, Sackett; in collaboration with Barr, Jin, Lafer, Prasad, Samelson, Smith

Many critical biological functions involve changes in the composition and shape of cellular membrane components. For example, protein trafficking in eukaryotic cells generally involves small tubulovesicular entities whose morphologies are linked to the binding of specific coat proteins. We have been investigating how the biogenesis of such vesicles is mediated by coat mechanics that are dependent on the molecular properties of coat constituents. Receptor-mediated endocytosis, for example, occurs through the formation of vesicles that are surrounded by polyhedral, cage-like structures assembled from a three-legged heteropolymer known as a clathrin triskelion. Work previously carried out by our group indicated that coats containing only clathrin and the principal clathrin-binding proteins (AP2 complexes) are unlikely to bend portions of a typical plasma membrane into small vesicles with a size similar to that of clathrin-coated vesicles (CCVs). To extend this work, we recently developed a new method, based on atomic force microscopy (AFM), to determine the mechanical rigidity of isolated intact CCVs. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM compressive force. We estimate that the bending rigidity of these vesicles is approximately 20 times that of either the outer clathrin cage or inner vesicle membrane, implying that the intermediate protein layer is normally rather flexible and that the binding of molecules that change coat rigidity might play a role in vesicle destabilization.

Another facet of our study relates to increasing evidence that signaling by membrane lipids, in particular 3′ phosphoinositides (3′PIs), is involved in clathrin-mediated vesicle formation as well as in other membrane transformations underlying cell function. The experimentally most thoroughly studied of the latter is chemotactic gradient sensing in immune and amoeboid cells, where external chemical stimuli induce cytoskeletal changes giving rise to directed cell locomotion. We have employed mathematical and computational methods to construct a biochemical network for PI signaling in chemotactic cells that includes actions of PI kinases, PI phosphatases, small g-proteins, and phosphatidic acid production. The network interactions contain coupled feedback and feedforward loops that can lead to regulated responses that act as switches. By allowing for translocation of molecules from cytosol to membrane that couple responses at distant points on the cell surface, the model demonstrates a range of gradient-sensing mechanisms and captures such characteristic behaviors as strong polarization in response to static gradients, adaptation to different mean levels of stimulus, and plasticity in response to changing gradients. We recently extended this earlier analysis to a study of the stability of polarized steady-state solutions. We find that if a cell is polarized in a background gradient, it will be most sensitive to transient point-source stimuli lying within a range of 40-80 degrees with respect to its polarization axis. The result can lead to zigzag movements of cells in chemotactic gradients, as noted under certain experimental conditions.

Finally, we have developed a method based on a modification of total internal reflection fluorescence (TIRF) microscopy that allows us to study the movement of signaling complexes at a cell surface. We have applied this technique to a study of T-cell antigen receptors, permitting estimates of the lifetimes of the associated receptor constituents.

Tubulin polymers and cytoskeletal organization

Sackett, Boukari; in collaboration with Li, Pannell, Schuck, Watts, Werbovetz

Tubulin polymers are central to various critical cell functions, including mitosis, intracellular transport, and maintenance of cell morphology and cell motility. We investigate the ability of small molecules to affect the formation of microtubules (MTs) and other cytoskeletal structures. Small molecules that alter MT integrity and/or dynamics can disrupt intracellular trafficking and change the physical properties of the cytoplasm. We aim to understand in detail the interactions of MTs with antimitotic peptide natural products from marine sources as well as with compounds produced by knowledge-directed synthesis. In particular, we have been studying how these materials induce MT subunits to assume unusual and characteristic ring shapes. We examined the structural and dynamic properties of these ring polymers by analytical ultracentrifugation, cryoelectron microscopy, fluorescence correlation spectroscopy, and protease mapping. The high stability and uniformity of the rings, as revealed by our studies, have led us to attempt their crystallization to achieve atomic resolution of their structures. After discovering an analogue of thalidomide that stabilizes microtubules, we also are examining the effects on microtubule polymerization of synthetic analogues of thalidomide and combretastatin A.

In addition, we are seeking to identify small molecules that avidly attach to parasite tubulin but do not bind to mammalian tubulin. Although tubulin has been well conserved during evolution, species differences do exist. Hence, for example, several molecules can target yeast rather than mammalian tubulin. We are looking for molecules that will similarly target Leishmania, which causes an important group of human diseases. In this regard, we have identified several small molecules that show promise as selective binding agents for Leishmania tubulin; they prevent parasite multiplication inside human macrophage cells. In addition, we have purified tubulin from cultured Leishmania and have characterized it for use in drug screening.

Polymer networks and structured media

Galanis, Michelman-Ribeiro, Boukari, Sackett, Nossal; in collaboration with Bansil, Harries, Losert, Margolis

Polymer networks are important elements of many biological materials, such as extracellular matrix, the cell cytoskeleton, mucus, the vitreous of the eye, sinovial fluid, and microbial biofilms. Many of the physical properties of these complex viscoelastic materials are not yet well understood. We thus have undertaken basic studies to illuminate general behaviors of such systems. For example, we recently investigated how polyelectrolyte gels reorganize in the presence of strong pH gradients. When unbuffered, finite-sized, agarose gels are subject to electric fields that induce electrolysis, we find that strong pH gradients are established across the gels and migrate in accordance with mathematical predictions of a continuum electrodiffusion model. By using small-angle light scattering, we have established that, as the electrophoretic fronts meet, gel domains arise that are oriented perpendicularly to the field. We also have been using fluorescent correlation spectroscopy to study the migration of particles within soft gels. We are currently using information from our studies in investigations of the physical properties of mucus, particularly with respect to how the physical state of mucus affects the penetration of macromolecules and viruses.

We have also investigated in what way physical boundaries affect spatial patterns set up by concentrated ensembles of rod-shaped granular materials, finding that, if confined to quasi-two-dimensional containers, the rods self-organize and show a density-dependent isotropic-nematic structural transition. A continuum theory of elastic energy explains the complex patterns that emerge as a result of competition between steric rod-rod interactions and interactions of the rods with the walls of the container. Recently, we have been studying the effects of "molecular crowders" on the spatial ordering of the rods.

COLLABORATORS

Rama Bansil, PhD, Boston University, Boston, MA
Valerie Barr, PhD, Laboratory of Cellular and Molecular Biology, NCI, Bethesda, MD
Daniel Harries, PhD, Hebrew University, Jerusalem, Israel
Ferenc Horkay, PhD, Laboratory of Integrative and Medical Biophysics, NICHD, Bethesda, MD
Albert J. Jin, PhD, Division of Biomedical Engineering and Physical Science, ORS, Bethesda, MD
Susan Krueger, PhD, Center for Neutron Research, NIST, Gaithersburg, MD
Eileen Lafer, PhD, University of Texas Southwestern Medical Center, San Antonio, TX
Pui-Kai Li, PhD, Ohio State University, Columbus, OH
Wolfgang Losert, PhD, University of Maryland, College Park, MD
Leonid Margolis, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
Lewis Pannell, PhD, Laboratory of Bioorganic Chemistry, NIDDK, Bethesda, MD
Khondury Prasad, PhD, University of Texas Southwestern Medical Center, San Antonio, TX
Lawrence E. Samelson, MD, Laboratory of Cellular and Molecular Biology, NCI, Bethesda, MD
Peter Schuck, PhD, Division of Biomedical Engineering and Physical Science, ORS, NIH, Bethesda, MD
Paul Smith, PhD, Division of Biomedical Engineering and Physical Science, ORS, NIH, Bethesda, MD
Norman Watts, PhD, Laboratory of Structural Biology Research, NIAMS, Bethesda, MD
Karl A. Werbovetz, PhD, Ohio State University, Columbus, OH

For further information, contact rjn@helix.nih.gov.