CELL BIOPHYSICS
, Head, Section on Cell Biophysics
Dan Sackett, PhD, Staff Scientist
Hacene Boukari, PhD, Senior Fellow
Jennifer Galanis, MD, Postdoctoral Fellow
Peter Krsko, PhD, Postdoctoral Fellow
Matt Ferguson, MS, Predoctoral Fellow
Ariel Michelman-Ribeiro, PhD, 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 improve our 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 thus have constructed specialized fluorescence-based optical instrumentation to study dynamical 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.
Biophysical methods and models
Boukari, Ferguson, Michelman-Ribeiro, Nossal, Sackett; in collaboation with Brichacek, Horkay, Krueger, Lafer, Margolis, Prasad, Schuck
We continue to expand the use of physics-based methodologies to study aspects of complex biological phenomena. For example, we develop new analytical methods based on fluorescence correlation spectroscopy, Fourier transform analysis, and quantitative microscopy to study biological structure and behavior. In particular, we have devised new techniques that make it possible to examine the movement of particles—varying in size from small metabolites to viruses—through concentrated polymer solutions and dense, interconnected polymer matrices. Initially, our studies used models of non-biological origin in order to establish the physical basis of the methods. Measurements indicated that the diffusion of materials through a polymer gel depends not only on the polymer concentration but also on the cross-link density of the matrix. We are now using similar techniques to examine the movement of antibodies and viruses through vaginal secretions. In the latter case, the goals are to understand how HIV and other viruses involved in sexually transmitted diseases penetrate cervical mucus and other protective barriers to reach the cells that they infect.
In another study, we used novel computer-based structural modeling, combined with dynamic light scattering (DLS), static light scattering (SLS), and small-angle neutron scattering (SANS), to examine conformations of clathrin triskelia in solution. Clathrin is a major protein involved in receptor-mediated endocytosis (RME), a process whereby eukaryotic cells take up growth factors and metabolites and regulate surface-bound receptors for those materials. The process involves the formation of protein-coated membrane vesicles whose biogenesis is not yet fully understood. To develop physical insights into the formation of clathrin-containing coats, we needed to determine if and how clathrin triskelia (three-legged complexes of clathrin heavy chains and clathrin light chains) change their shape when they leave solution to assemble into coat-associated cages. We have shown that the triskelia are puckered when free, but with a somewhat different conformation than when incorporated into a reconstituted spherical clathrin basket. Our studies were the first to provide this information. We also developed a novel method, based on SANS, to assess the mechanical flexibility of the triskelia. The work derives, in part, from our earlier investigations on nanoscopic tubulin rings formed in the presence of certain small peptides considered antimitotic agents for cancer therapy.
Boukari H, Sackett DL, Schuck P, Nossal R. Single-walled tubulin ring polymers. Biopolymers 2007;86:424-36.
Ferguson ML, Prasad K, Sackett DL, Boukari H, Lafer E, Nossal R. Conformation of a clathrin triskelion in solution. Biochemistry 2006;45:5916-22.
Michelman-Ribeiro A, Horkay F, Nossal R, Boukari H. Probe diffusion in aqueous poly(vinyl alcohol) solutions studied by fluorescence correlation spectroscopy. Biomacromolecules 2007;8:1595-600.
Svitel J, Boukari H, Ryk DV, Wilson RC, Schuck P. Probing the functional heterogeneity of surface binding sites by analysis of experimental binding traces and the effect of mass transport limitation. Biophys J 2007;92:1742-58.
Biophysics of supramolecular assembly
Many critical biological functions involve the assembly of supramolecular structures that are large oligomers of specialized proteins. For example, as previously mentioned, 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 a clathrin triskelion. We have been developing physical theories to understand how such vesicles (clathrin-coated vesicles, or CCVs) arise. Several observations point to the importance of macromolecular mechanical properties in determining the sizes of the cages, and we have conducted a series of studies to infer quantitative values of rigidity parameters of clathrin structures. For example, we recently developed a new method, based on atomic force microscopy (AFM), to determine the mechanical rigidity of isolated intact CCVs. We found that the static bending rigidity of a coated vesicle is in reasonable agreement with values of clathrin cage rigidity determined by other means. We are currently attempting to extend these measurements to assess the viscoelastic, time-dependent behavior of the vesicles. We also have devised a scheme, based on SANS and molecular modeling, that allows us to assess the mechanical properties of triskelia suspended in solution, thereby enabling direct determination of quantities that previously could be deduced only by somewhat oblique methodology. Recent results substantiate the earlier inferences, establishing that triskelia can bend as they are integrated into polyhedral coats of various sizes and that, furthermore, the clathrin lattice by itself is unlikely to be strong enough to cause vesicles to form with the high curvature noted in cell-biological observations.
Another project focuses on obtaining a basic understanding of how physical boundaries influence spatial patterns that arise in concentrated ensembles of rod-shaped objects such as microtubules, amyloid plaques, and similarly shaped biological assemblies. We constructed a biomimetic analogue, composed of small hard rods confined within an enclosure of adjustable size and shape, and subjected the analogue to mechanical shaking to mimic thermal excitation. The objective was to study the effects of steric interference between for example, microtubules independent of other intermolecular interactions. We found that when the rods are confined to containers whose dimensions are of the same order of magnitude as the lengths of the rods, conditions permit the rods to self-organize and experience a density-dependent isotropic-nematic structure transition. This model pertains at an elemental level to microtubule involvement in cell division. Recently, to investigate the role of cytoplasmic factors that act as molecular crowders, we added small spheres to the ensemble of objects undergoing thermal excitation. We noted new rod structures that, under certain conditions, differ dramatically from those seen in the absence of the spheres. In our earlier work, we developed a continuum mathematical theory that explains how the observed patterns result from a competition between steric rod-rod interactions in the bulk and interactions of the rods with the container walls. We are currently developing an extension of that analysis to include the effects of the crowders.
Galanis J, Harries D, Sackett DL, Losert W, Nossal R. Spontaneous patterning of confined granular rods. Phys Rev Lett 2006;96:028002.
Jin AJ, Prasad K, Smith PD, Lafer EM, Nossal R. Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy. Biophys J 2006;909:3333-44.
Tubulin polymers and cytoskeletal organization
Tubulin polymers are central to various critical cell functions, including mitosis, intracellular transport, maintenance of cell morphology, and cell motility. Our project focuses on 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 and with compounds produced by knowledge-directed synthesis.
We also 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 exist. Hence, for example, several molecules can target yeast rather than mammalian tubulin. We are looking for molecules that will similarly target Leishmania, the infectious cause of an important group of human diseases. To advance our studies, we have purified and characterized tubulin from a non-pathogenic species of Leishmania, thereby providing a more ready supply of the needed protein than was available from the pathogenic strain and facilitating the screening of potential drugs. We also cloned the genes for both subunits of tubulin from Leishmania and are attempting to produce recombinant Leishmania tubulin in bacteria. If we succeed, we will be able to produce tubulin free of the abundant post-translational modifications (PTMs) present in the natural protein and generate the first successful bacterial production of recombinant tubulin.
In addition to our in vitro studies, we have investigated the intracellular function of MTs in terms of both PTM of tubulin and the functional role of MT arrays in intracellular transport. PTMs such as acetylation play a role in regulating diverse protein systems in the cell; prominent examples are tubulin in the cytoplasm and histones in the nucleus. Both are affected by small molecule histone deacetylase inhibitors currently undergoing clinical development. We have continued to investigate the action of these compounds on chromatin, MTs, and the interactions between them that are essential to mitosis; we have built on our previous demonstration that these compounds cause mitotic arrest as a consequence of MTs’ failure to bind properly with chromosomes during early mitosis.
Signaling between the cytoplasm and the nucleus often occurs via MT-mediated transport. We have previously shown that such signaling is a necessary part of the p53-mediated response to DNA damage and that it occurs through movement of p53 to the nucleus on MTs by the motor protein dynein. Further research has demonstrated that assembly of p53 oligomers precedes binding to dynein. Mutations, including mutations found in patient samples, that compromise oligomerization result in cytoplasmic sequestration of p53 and abolition of the p53 response.
George TG, Johnsamuel J, Delfín DA, Yakovich A, Mukherjee M, Phelps MA, Dalton JT, Sackett DL, Kaiser M, Brun R, Werbovetz KA. Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides. Bioorg Med Chem 2006;14:5699-710.
Piekarz RL, Sackett DL, Bates SE. Histone deacetylase inhibitors and demethylating agents: clinical development of histone deacetylase inhibitors for cancer therapy. Cancer J 2007;13:30-9.
Trostel SY, Sackett DL, Fojo T. Oligomerization of p53 precedes its association with dynein and nuclear accumulation. Cell Cycle 2006;5:2253-9.
Yakovich AJ, Ragone FL, Alfonzo JD, Sackett DL, Werbovetz KA. Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening. Exp Parasitol 2006;114:289-96.
Complex systems biophysics
We use advanced physical and mathematical methods to understand the biophysics of complex cellular processes. Phenomena recently under study include chemotactic gradient sensing in eukaryotic cells, the stochastic biogenesis of coated vesicles involved in endocytosis and other intracellular transport processes, and the structural organization of multicellular biofilms arising from the attachment of prokaryotes to surfaces in nutrient-rich environments. Our studies are relevant to basic cell-biological processes as well as to disease processes and normal and abnormal tissue development. Each requires the integration of several complicated processes that use information obtained through reductionist studies. However, here we focus on behaviors emerging from both synergistic and competitive interactions.
Chemotaxis, the spatially directed cell response to gradients of chemical signals, is an important element in critical processes such as wound healing, immune surveillance, tissue development, angiogenesis, and the creation of connections between nerve cells. The first step in these processes, namely, gradient detection, has long been a subject of active investigation. We have devised a mathematical model, based on nonlinear reaction-diffusion equations for concentrations of 3¢-phosphoinositides, PI3-kinases, and PTEN phosphatases, that captures the three major behaviors of these biochemical species as observed in the chemotactic response of Dictyostelium and neutrophils: establishment of cell polarity in shallow spatial gradients of stimulus; adaptation to changes in uniform background levels of signaling ligand; and the ability of a cell to follow rapidly the movements of an excitatory chemical source. In contrast with other treatments of this problem, our study explicitly incorporates plausible biochemical mechanisms, allowing us to gain insights into how molecular processes mediate the chemotactic response. A recent extension of our earlier work now has provided a way to infer the angle dependence of the sensitivity of an already polarized cell on the location of the source, providing an explanation for the zig-zag motions occasionally observed when a neutrophil travels toward a target.
In addition to their involvement in gradient sensing, 3¢ phosphoinositides are implicated in the biogenesis of clathrin-coated and other endocytic vesicles. We have constructed a complex, multi-element model of receptor-mediated endocytosis that encompasses cargo recognition, phosphoinositide metabolism, and clathrin coat formation and dissolution. The analysis demonstrates how the inter-related kinetic elements of these processes determine whether an endocytic vesicle will form. Not only does the model explain how vesicle biogenesis is triggered by, for example, the binding of ligands to receptors at specific sites, but it also can rationalize the observed probabilistic quality of cell response in the presence of a stimulus.
Another area of complex systems biology currently under investigation pertains to bacterial biofilms, which are surface-attached communities of microorganisms that express a polymer coating—the extracellular polymeric substance (EPS)—that protects the attached bacterial colonies from antimicrobial agents. Biofilms are ubiquitous in the natural and technologically modified worlds, yet little is understood about their formation and viability. In human disease, many bacterial pathogens form biofilms that resist destruction, giving rise to intractable and dangerous infections. We have focused on measuring the mechanical and transport properties of the EPS as a function of environmental parameters such as pH and externally induced shear forces and are working to identify factors that affect the flow of antibiotics within a biofilm and to understand how the EPS mediates the activity of immune cells. We are also investigating how biofilms, which are amenable to external manipulation, may serve as rudimentary models for studying the growth and regeneration of even more complex cell communities. To characterize the mechanical properties of the EPS, we have developed methods involving AFM that allow us to take into account the spatial heterogeneity of the colonies. We have found that the soft, hydrated EPS gel, which consists mainly of polysaccharides, proteins, and nucleic acids carrying labile charges, softens and stiffens according to the proton concentration in the surrounding environment.
Nossal R. Stochastic trigger for clathrin-coated vesicle biogenesis. Proc SPIE 2007;6602:J1-12.
Skupsky R, Losert W, Nossal R. Distinguishing modes of eukaryotic gradient sensing. Biophys J 2005;89:2806-23.
Skupsky R, McCann C, Nossal R, Losert W. Bias in the gradient-sensing response of chemotactic cells. J Theor Biol 2007;247:242-58.
COLLABORATORS
Susan Bates, MD, Medical Oncology Branch, NCI, Bethesda, MD
Beda Brichacek, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Tito Fojo, MD, Medical Oncology Branch, NCI, Bethesda, MD
Daniel Harries, PhD, Hebrew University, Jerusalem, Israel
Ferenc Horkay, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Albert J. Jin, PhD, Division of Biomedical Engineering and Physical Science, ORS, Bethesda, MD
Paul Kolenbrander,PhD, Oral Infection and Immunity Branch, NICDR, 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
Leonid Margolis, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Rob Palmer, PhD, Oral Infection and Immunity Branch, NICDR, 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, NIH, Bethesda, MD
Ron Skupsky, PhD, University of California Berkeley, Berkeley, CA
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
Al Yergey, PhD, Program in Physical Biology, NICHD, Bethesda, MD
For further information, contact nossalr@mail.nih.gov.
