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 interactions and transformations of supramolecular cellular assemblages such as protein-coated endocytic vesicles, metabolic signaling complexes, and cytoskeletal structures and networks. Thus, for example, we have constructed specialized fluorescence-based optical instrumentation to study dynamic processes and have used advanced electromagnetic scattering techniques to examine structures on nanoscopic length scales. We also develop mathematical models of specific aspects of supramolecular and cellular behavior.
Fluorescence correlation spectroscopy and Fourier imaging (scattering) techniques
Boukari, Nossal, Michelman-Ribeiro, Sackett; in collaboration with Bansil, Horkay, Krueger, Schuck
We have been developing fluorescence correlation spectroscopy (FCS) as a tool to examine properties of supramolecular biological assemblies, with particular emphasis on the movement of probe molecules within hydrogels and other complex polymer networks. For example, we used FCS to obtain quantitative measures of the diffusion time of fluorescent TAMRA (tetramethylrhodamine) molecules in poly(vinyl alcohol) (PVA) solutions and gels prepared at various polymer concentrations and cross-link densities. The measurements indicated that the diffusion rate was affected not only by the polymer concentration but also by the cross-link density of the gel. For both solutions and gels, the diffusion rate, normalized to the rate in pure water, appeared to decrease linearly with the polymer concentration. Below the gelation threshold (approximately 3 percent PVA w/v), diffusion rates did not change with the addition of cross-linkers, but above the threshold movement of the probe through the gels was slower than in the corresponding polymer solutions. Interestingly, we found that diffusion of the probe particles in the gels was strongly correlated with network elasticity. We have also been using FCS and dynamic light scattering 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 analytic ultracentrifugation of the rings, we corroborated theories and computational code that provide values of hydrodynamic coefficients of particles of complex geometry. We have used the insights gained from these investigations to guide studies of the shapes of clathrin triskelions in solutions of different pH and salt concentrations.
Boukari H, Nossal R, Sackett DL, Schuck P. Hydrodynamics of nanoscopic tubulin rings in dilute solution. Phys Rev Lett 2004;93:098106.
Boukari H, Sackett DL, Nossal R. Probing the hydrodynamic behavior of drug-induced tubulin rings by fluorescence correlation spectroscopy. Macromol Symp 2005;227:211-220.
Michelman-Ribeiro A, Boukari H, Nossal R, Horkay F. Fluorescence correlation spectroscopy study of probe diffusion in poly(vinyl alcohol) solutions and gels. Macromol Symp 2005;227:221-230.
Michelman-Ribeiro A, Boukari H, Nossal R, Horkay F. Structural changes in polymer gels probed by fluorescence correlation spectroscopy. Macromolecules 2004;37:10212-10214.
Membrane transformations underlying cell function
Nossal, Ferguson, Boukari, Sackett; in collaboration with Jin, Lafer, Muthukumar, Prasad, 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 the production of small tubulovesicular entities linked to the binding of specific coat proteins. In particular, receptor-mediated endocytosis occurs through the formation of vesicles that are surrounded by polyhedral, cage-like structures assembled from a three-legged heteropolymer known as the clathrin triskelion. We have been investigating the biogenesis of such clathrin-coated vesicles. To this end, we used a variety of physical methods to infer how coat mechanics influences vesicle formation and to understand how environmental factors and the binding of ancillary proteins (e.g., AP2 complexes) might mediate clathrin lattice assembly. Our previous work indicated that coats containing only clathrin and APs are unlikely to bend portions of a typical plasma membrane into small vesicles of a size similar to that of clathrin-coated vesicles.
To extend our work, we recently developed a new method, based on atomic force microscopy, to determine the mechanical rigidity of intact clathrin-coated vesicles (CCVs). Such direct measurement of the mechanical properties of CCVs allows us to infer the mechanical nature of the protein layer that links the outer clathrin cage to the inner lipid shell of a typical CCV. We found that the protein layer is relatively flexible, implying that changes in coat rigidity can modulate vesicle biogenesis. In a related study, we used static and dynamic light scattering, combined with computer-based structural modeling, to examine the conformations of triskelions in solution. We found that the triskelions have a geometric form close to that discerned elsewhere by cryoelectron microscopy of reconstituted clathrin cages. Hence, when inserted into cages and coats, clathrin triskelia probably adjust to the geometry of the coat at the cost of only relatively small strain energy. In addition, it is clear that the binding of clathrin-associated proteins to phosphoinositides (PIs) plays an important role in vesicle formation, perhaps by inducing curvature changes in the lipid membrane. Evidence is gathering to suggest that signaling by membrane lipids, in particular 3´ phosphoinositides (3´ PIs), is involved in this and other membrane transformations underlying cell function. The best studied of these phenomena is gradient sensing in immune and amoeboid cells, in which information about external chemical stimuli induces cytoskeletal changes, giving rise to directed cell locomotion.
We have employed mathematical and computational methods to construct and examine a biochemical network for PI signaling that includes actions of PI kinases and phosphatases, small G proteins, and phosphatidic acid production. The network interactions include coupled feedback and feed-forward 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, we have shown how different magnitudes of system parameters can result in characteristically different cellular behaviors that mirror environmental changes.
Jin AJ, Prasad K, Smith PD, Lafer EM, Nossal R. Measuring clathrin coated vesicle elasticity via atomic force microscopy. Biophys J (in press).
Nossal R. Assembly of clathrin baskets. Macromol Symp 2005;219:1-8.
Nossal R. Mechanical aspects of clathrin cage formation. Macromol Symp 2005;227:17-26.
Skupsky R, Losert W, Nossal RJ. Distinguishing modes of eukaryotic gradient sensing. Biophys J 2005;89:2806-2823.
Tubulin polymers and cytoskeletal organization
Sackett, Nossal, Boukari, Galanis; in collaboration with Bates, Fojo, Krueger, Losert, Pannell, Werbovetz
Tubulin polymers are centrally involved in various key cell functions, including mitosis, intracellular transport, establishment and maintenance of cell morphology, and cell motility. In such processes, tubulin occurs prominently as microtubules; the disturbance of microtubules usually results in dramatic modification of cell behavior. However, tubulin polymers appear in many forms, depending on environmental factors such as pH, salt concentrations, temperature, and the presence of small organic molecules that mediate tubulin-tubulin interactions. For some time, we have been investigating how these factors affect resultant supramolecular tubulin structure. In particular, we have undertaken studies aimed at establishing how certain marine natural products, including cryptophycin and dolastatin, induce single-walled tubulin rings. Recently, we used FCS to study the stability of the rings at low tubulin concentration (see above). In collaboration with other investigators and with the goal of identifying drugs active against Leishmania donovani and related organisms, we have also studied the ability of certain herbicide derivatives to target kinetoplastid parasite tubulin selectively. We also have begun several studies of biomimetic systems aimed at understanding physical aspects of the self-organization of microtubules into large-scale arrays. For example, we have investigated the ordering of rod-like particles constrained to small enclosed spaces and have noted that, despite an interplay between local steric ordering and orientation along the walls of the container, nematic-like ordering occurs as particle density increases.
Kim YJ, Pannell LK, Sackett DL. Mass spectrometric measurement of differential reactivity of cysteine to localize protein-ligand binding sites. Application to tubulin-binding drugs. Anal Biochem 2004;332:376-383.
Poruchynsky MS, Kim J-H, Nogales E, Annable T, Loganzo F, Greenberger LM, Sackett DL, Fojo T. Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analog, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability. Biochemistry 2004;43:13944-13954.
Robbins AR, Jablonski SA, Yen TJ, Yoda K, Robey R, Bates SE, Sackett DL. Inhibitors of histone deacetylases alter kinetochore assembly by disrupting pericentromeric heterochromatin. Cell Cycle 2005;4:717-726.
Sackett DL. Ring polymers of tubulin induced by binding of natural antimitotic peptides. Macromol Symp 2005;219:9-16.
Polymer networks and structured media
Galanis, Michelman-Ribeiro, Sackett, Nossal; in collaboration with Bansil, Harries, Losert
Polymer networks are important elements of many biological materials. Examples include extracellular matrix, the cell cytoskeleton, mucus, the vitreous humor of the eye, sinovial fluid, biofilms, and so forth. In addition, hydrogels have found several applications in biotechnology. Many of the physical properties of these complex viscoelastic materials are not yet well understood. Thus, we undertake basic studies that, even if lacking immediate biological application, can illuminate general behaviors of such systems. For example, we recently investigated how polyelectrolyte gels reorganize in the presence of strong pH gradients. When we subject unbuffered, finite-sized agarose gels 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 fronts meet, gel domains arise that are oriented perpendicularly to the field. We also investigated how physical boundaries affect spatial patterns set up by concentrated ensembles of rod-shaped granular materials, finding that, if confined to quasi-2D 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 in the bulk and interactions of the rods with the walls of the container.
Galanis J, Harries D, Sackett DL, Losert W, Nossal R. Spontaneous patterning of confined granular rods. Phys Rev Lett (in press).
Michelman A, Nossal R, Morris R, Lange S, Kuo C-S, Bansil R. Electrolysis induces pH gradients and domain orientation in agarose gels. Phys Rev E (in press).
Nossal R. Zoetic polymers. Biophys Chem 2004;112:219-222.
COLLABORATORS
Susan Bates, MD, Cancer Therapeutics Branch, NCI, Bethesda, MD
Rama Bansil, PhD, Boston University, Boston, MA
Tito Fojo, MD, PhD, Cancer Therapeutics Branch, NCI, Bethesda, MD
Daniel Harries, PhD, Laboratory of Physical and Structural Biology, NICHD, Bethesda, MD
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
Wolfgang Losert, PhD, University of Maryland, College Park, MD
Muragappan Muthukumar, PhD, University of Massachusetts, Amherst, MA
Lewis Pannell, PhD, Laboratory of Bioorganic Chemistry, NIDDK, Bethesda, MD
Khondury Prasad, PhD, University of Texas Southwestern Medical Center, San Antonio, TX
Peter Schuck, PhD, Division of Biomedical Engineering and Physical Science, ORS, Bethesda, MD
Paul Smith, PhD, Division of Biomedical Engineering and Physical Science, ORS, Bethesda, MD
Karl A. Werbovetz, PhD, Ohio State University, Columbus, OH
For further information, contact rjn@helix.nih.gov.