Cell Biophysics

Ralph Nossal, PhD, Head, Section on Cell Biophysics
Dan Sackett, PhD, Staff Scientist
Hacene Boukari, PhD, Senior Fellow
Jennifer Galanis, MD, Guest Researcher
Peter Krsko, PhD, Postdoctoral Fellow
Adrian Begaye, BA, BS, Postbaccalaureate Fellow

We study 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. We develop and apply novel methodologies based on mathematical and physical principles in order to foster a better understanding of such phenomena. We focus on 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 dynamic supramolecular processes and use advanced electromagnetic scattering techniques to examine structures on nanoscopic length scales. We have also developed mathematical models of specific aspects of cellular behavior.

Biophysical methods and models

Boukari, Nossal, Sackett; in collaboration with Michelman-Ribeiro, Horkay, Silva, Brichacek, Margolis, Gandjbakhche, Riley

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 (FCS), Fourier transform analysis, and quantitative microscopy to study biological structure and behavior. In particular, we devised new techniques that permit us 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 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. Recently, to develop a fuller comprehension of underlying physical mechanisms, we focused on discerning how the swelling or shrinking of a cross-linked hydrogel affects the translation of embedded nanoparticles. The knowledge gained should ultimately lead to, for example, a deeper understanding of the behavior of extracellular matrix material in edematous tissue. We are now employing related techniques to examine the movement of antibodies and viruses through vaginal secretions. In the case of viruses, our goal is to understand how HIV and other viruses involved in sexually transmitted diseases penetrate cervical mucus and other protective barriers to reach the cells they infect. Particle-tracking analysis of individual virions indicates that most slow down over 100-fold when compared with their movement in water and that typical diffusion does not drive the virions’ translocation. Rather, a major factor is relaxation of the matrix following mechanical perturbations such as stretching and twisting of a sample. Such relaxation gives rise to measured, ensemble-averaged displacements of virions, demonstrating a combination of anomalous diffusion and occasional jumps influenced by microenvironment properties sensed by individual virus particles.

The above phenomena occur in relatively dense media, where significant light scattering from the surrounding matrix might cause problems. In FCS measurements, for example, analysis requires reliable determination of the illuminated volume, but the incident beam might be distorted by multiple scattering. Hence, we carried out Monte Carlo simulations to examine the effects of scattering induced by a crowded solution of spherical nanoparticles and found that, as the concentration or size of the particles increases, the beam-spot broadens and its focal point is shifted toward the direction of the source. We also found a concomitant reduction of the local laser beam flux. Such effects may need to be accounted for when using FCS to investigate optically dense tissue. One goal of these studies is to provide metrics that enable us to make quantitative corrections where appropriate.

Boukari H, Silva CS, Nossal R, Horkay F. Monitoring nanoprobe diffusion in osmotically-stressed hydrogels. Mater Res Soc Symp Proc 2008;1060E:LL07-13.
Michelman-Ribeiro A, Horkay F, Nossal R, Boukari H. Probe diffusion in aqueous poly(vinyl alcohol) solutions studied by fluorescence correlation spectroscopy. Biomacromolecules 2007;8:1595-1600.

Clathrin lattice and other supramolecular assembly

Galanis, Boukari, Sackett, Nossal; in collaboration with Ferguson, Lafer, Prasad, Krueger, Harries, Schuck

Many biological functions involve the assembly of large supramolecular structures that, at first approximation, are large oligomers of specialized proteins. Receptor-mediated endocytosis, however, is a special case that involves the formation of vesicles surrounded by closed polyhedral, cage-like structures assembled from a three-legged heteropolymer, which is a triskelion composed of three clathrin heavy/light chain complexes joined at a common hub. In developing physical theories to understand how such vesicles (clathrin-coated vesicles, or CCVs) arise, we have quantitatively analyzed previously published data and have acquired our own data. Most recently, 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. We were interested in determining if and how clathrin triskelia change their shape when they leave solution to assemble into coat-associated cages. We showed that triskelia are puckered when free in solution, although they exhibit a somewhat different conformation than when incorporated into a reconstituted clathrin basket, suggesting that the mechanical properties of triskelia indeed must be taken into account. Therefore, we developed a novel method based on SANS to assess the flexibility of the triskelia, permitting us to determine quantities that previously could be deduced only by somewhat indirect methodology. Results substantiate our earlier hypotheses and inferences, establishing that triskelia are semiflexible polymeric structures capable of bending when integrated into polyhedral coats of various sizes and shapes. In addition, the clathrin lattice by itself is unlikely to be sufficiently strong to cause vesicles to form with the high curvature noted in cell-biological observations. We are now applying the knowledge gained from these and related studies to investigate clathrin pit formation at the plasma membrane, with a focus on discovering how the binding of clathrin to accessory proteins such as AP-2 and AP-180 influences clathrin coat nucleation.

In another, somewhat more abstract project, we set out to study the effects of steric interference between microtubules apart from other intermolecular interactions. We aim to obtain 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 enclosures of various sizes and shapes and subjected them to mechanical shaking to mimic thermal excitation. 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. Recently, to investigate the role of factors that act as molecular crowders in processes such as the formation of protein aggregates on membrane surfaces, we added small spheres to such rod assemblies. Given that macroscopic models can provide information about the assembly of complex molecular structures not easily obtained by other means, we found that, for sufficiently low rod densities, “vibrofluidized” rods self-assemble in a “solution” of sphere crowders to form linear polymer-like structures. We also found that Monte Carlo simulations show similar patterns in thermally equilibrated binary mixtures. Unlike the known equilibrium phases for rod-sphere mixtures in three dimensions, our two-dimensional simulations and experiments show finite-sized aggregates, indicating that lower dimensionality prevents true phase separation and that thermodynamic depletion interactions create an effective attractive force between the rods.

Boukari H, Sackett DL, Schuck P, Nossal R. Single-walled tubulin ring polymers. Biopolymers 2007;86:424-436.
Ferguson ML, Prasad K, Boukari H, Sackett DL, Krueger S, Lafer EM, Nossal R. Clathrin triskelia show evidence of molecular flexibility. Biophys J 2008;95:1945-1955.

Tubulin polymers and cytoskeletal organization

Sackett, Boukari, Begaye; in collaboration with Bezrukov, Rostovtseva, Yergey, Bates, Fojo, Park, Li, Werbovetz, Fecik, Schriemer

We study both fundamental and applied aspects of tubulin biology. Tubulin polymers are central to various critical cell functions, including mitosis, intracellular transport, maintenance of cell morphology, cell motility, and perhaps mitochondrial function. We have devoted considerable effort to determining the ability of small molecules to alter the formation of microtubules (MT), which are the most rigid of the cytoskeletal polymers and thus are central to establishing non-spherical cell morphology. In addition, MTs have intrinsic polarity, and their cytoplasmic array provides the substrate for directional intracellular movement. Thus, small molecules that alter MT integrity and/or dynamics can alter the physical properties of the cytoplasm. We have been studying anti-mitotic peptides, often of marine origin, because they are among the most potent anti–MT agents known; moreover, several such peptides are available for study. In particular, certain peptides induce MT subunits to assume unusual ring-shaped supramolecular structures, which we have been studying by analytical ultracentrifugation, cryo-electronmicroscopy, fluorescence correlation spectroscopy, and protease mapping. We are also examining the effects on microtubule polymerization of synthetic analogues of thalidomide and combrestatin A and have demonstrated both the role of post-translational modifications by deacetylases and the effects of proteosome inhibitors on microtubule stability.

Recently, we identified several new modified peptides derived from the natural peptide tubulysin; these peptides show a range of anti-microtubule activities in assays with purified proteins as well as in cells. In addition, focusing on the natural compound peloruside, we examined a new type of microtubule stabilizer and demonstrated that an old chemotherapy agent, a nitrosourea, may indirectly affect microtubule stability by altering the activity of the protein stathmin. In additionally, in an effort to look for molecules that would target Leishmania, the infectious agent of an important group of human diseases, we are seeking to identify small molecules that do not bind well to mammalian tubulin but do bind to parasite tubulin. We have thus far identified several small molecules that bind to Leishmania tubulin preferentially over mammalian tubulin, preventing parasite multiplication inside human macrophage cells.

Finally, we have been studying the interactions between tubulin and supramolecular entities that affect metabolic activities of mitochondria—organelles that move along microtubules in mammalian cells. We examined the effect of tubulin binding on the function of the major mitochondrial outer membrane (MOM) channel protein VDAC¹ and found that nanomolar concentrations of tubulin cause closure of the channel. This closure, which requires the C-terminal tail peptides found on alpha and beta tubulin, is expected to restrict exchange of ADP and ATP across the MOM. Using isolated intact mitochondria, we confirmed the effect. Added tubulin reduces the oxygen uptake associated with added ADP as reflected in a nearly 100-fold increase in K_M for ADP. Our results show that tubulin can be involved in regulating respiration in mammalian cells by interacting with VDAC on the outer membrane of mitochondria.

Huzil JT, Chik JK, Slysz GW, Freedman H, Tuszynski J, Taylor RE, Sackett DL, Schriemer DC. A unique mode of microtubule stabilization induced by peloruside A. J Mol Biol 2008;378:1016-1030.
Liang XJ, Choi Y, Sackett DL, Park JK. Nitrosoureas inhibit the stathmin-mediated migration and invasion of malignant glioma cells. Cancer Res 2008;68:5267-5272.
Raghavan B, Balasubramanian R, Steele JC, Sackett DL, Fecik RA. Cytotoxic simplified tubulysin analogues. J Med Chem 2008;51:1530-1533.
Sackett DL. Antimicrotubule agents that bind covalently to tubulin. In: Fojo T, Teicher BA, eds. The Role of Microtubules in Cell Biology, Neurobiology, and Oncology. Humana Press, 2008;281-306.
Sackett DL , Ozbun L, Zudaire E, Wessner L, Chirgwin JM, Cuttitta F, Martínez A. Intracellular proadrenomedullin-derived peptides decorate the microtubules and contribute to cytoskeleton function. Endocrinology 2008;149:2888-2898.

Complex systems biophysics

Krsko, Sackett, Nossal; in collaboration with Skupsky, Losert, Kolenbrander, Palmer

We continue to use advanced physical and mathematical methods to understand the biophysics of complex cellular processes. The role of phosphoinositide metabolism in eukaryotic cell processes such as gradient sensing in chemotactic cells and the biogenesis of coated vesicles involved in endocytosis and other intracellular transport processes is one such phenomenon. We also study the structural organization of multicellular biofilms arising from the attachment of prokaryotes to surfaces, with a focus on the extracellular polymeric substance (EPS) that fills the space between bacterial colonies. Our studies are of basic interest but are also relevant to disease processes and normal and abnormal tissue development. Each study requires the integration of several complicated processes and relies on information obtained through reductionist studies while focusing on behaviors emerging from both synergistic and competitive interactions.

We previously devised a mathematical model—based on non-linear reaction-diffusion—that captures the major kinetic behaviors of the gradient sensing observed in the chemotactic response of amoeboid cells such as Dictyostelium and neutrophils. Among these behaviors are the establishment of cell polarity in shallow gradients of stimulus, adaptation to changes in uniform background levels of signaling ligand, and the ability of a cell to rapidly follow movements of an excitatory chemical source. A recent extension of our work has provided an explanation for the distinct zig-zag motions occasionally observed when a neutrophil travels toward a source of chemotaxins. In addition to their involvement in gradient sensing, 3′ phosphoinositides are implicated in the biogenesis of clathrin-coated and other endocytic vesicles. We are constructing a complex, multi-element model of receptor-mediated endocytosis that encompasses cargo recognition, phosphoinositide metabolism, and clathrin coat formation and dissolution. Our analysis, which demonstrates how interrelated kinetic elements of these processes determine whether an endocytic vesicle will form, will permit us to explain how vesicle biogenesis is triggered by, for example, the binding of ligands to receptors at specific sites and subsequent recruitment of clathrin-associated proteins. We are also interested in rationalizing the observed probabilistic quality of cell response in the presence of a stimulus.

We are currently investigating various characteristics of bacterial biofilms. These films are surface-attached communities of microorganisms that express a polymer coating (EPS ) that mediates material transport to the attached bacterial colonies and protects them from antimicrobial agents. Biofilms are ubiquitous in nature, yet little is known about their formation and viability. We do know that, in human disease, many bacterial pathogens form biofilms that resist destruction, causing serious difficulties for infected patients. We have focused on measuring the mechanical and transport properties of EPS as a function of environmental parameters such as pH and externally induced shear forces and have linked the parameter to morphological features of the films. To characterize the mechanical properties of EPS, we have developed methods involving atomic force microscopy that allow us to take into account the spatial distribution of bacterial colonies within the film. We found that the soft, hydrated EPS gel, which consists mainly of polysaccharides and other macromolecules carrying labile charges, softens and stiffens according to the proton concentration in the surrounding environment, concomitantly changing the mechanical properties of the colonies. Using scanning electron microscopy, we have developed an improved technique for morphological analysis of the biofilms that preserves the structure of these fragile and highly hydrated materials upon drying, hence revealing finer details of their in situ architecture and cellular adhesion. Finally, we fabricated a multichannel culture chamber from cast PDMS² that allows examination of biofilms with optical instruments as well as with direct contact instruments such as an atomic force microscope.

Nossal R. Stochastic trigger for clathrin-coated vesicle biogenesis. Proc SPIE 2007;6602:J1-J12.
Skupsky R, McCann C, Nossal R, Losert W. Bias in the gradient-sensing response of chemotactic cells. J Theor Biol 2007;247:242-258.

¹ voltage-dependent anion channel
² polydimethylsiloxane

Collaborators

Susan Bates, MD, Medical Oncology Branch, NCI, Bethesda, MD
Sergey Bezrukov, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Beda Brichacek, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Charles Eggleton, PhD, University of Maryland Baltimore County, Glen Burnie, MD
Robert A. Fecik, PhD, University of Minnesota, Minneapolis, MN
Matthew Ferguson, PhD, Centre de Biochimie Structurale, Montpellier, France
Tito Fojo, MD, Medical Oncology Branch, NCI, Bethesda, MD
Amir Gandjbakhche, PhD, Program in Physical Biology, NICHD, 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
Pui-Kai Li, PhD, Ohio State University, Columbus, OH
Wolfgang Losert, PhD, University of Maryland, College Park, MD
Leonid Margolis, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Ariel Michelman-Ribeiro, PhD, Semiconductor Electronics Division, NIST, Gaithersburg, MD
Rob Palmer, PhD, Oral Infection and Immunity Branch, NICDR, Bethesda, MD
John Park, MD, Surgical Neurology Branch, NINDS, Bethesda, MD
Khondury Prasad, PhD, University of Texas Southwestern Medical Center, San Antonio, TX
Jason Riley, PhD, University College London, London, UK
Tatiana Rostovtseva, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Dave Schriemer, PhD, University of Calgary, Calgary, Canada
Peter Schuck, PhD, Division of Biomedical Engineering and Physical Science, ORS, Bethesda, MD
Candida Silva, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Ron Skupsky, PhD, University of California Berkeley, Berkeley, CA
Karl A. Werbovetz, PhD, Ohio State University, Columbus, OH
Al Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact nossalr@mail.nih.gov.