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Cell Biophysics

Ralph Nossal, PhD
  • Ralph Nossal, PhD, Head, Section on Cell Biophysics
  • Dan Sackett, PhD, Staff Scientist
  • Anand Banerjee, PhD, Visiting Fellow
  • Silviya Zustiak, PhD, Postdoctoral Fellow
  • Adrian Begaye, BA, BS, Postbaccalaureate Fellow
  • Hacène Boukari, PhD, Guest Researcher
  • Jennifer Galanis, MD, Guest Researcher

We study elements of cell processes involved in signal transduction, protein trafficking, microtubule biology, and cell division. We focus on the origination and transformations of supramolecular cellular assemblages such as protein-coated endocytic vesicles, metabolic signaling complexes, and cytoskeletal structures. In our research, we develop and apply novel methodologies based on mathematical and physical principles. For example, we constructed specialized fluorescence-based optical instrumentation to study the dynamics of supramolecular processes and used advanced electromagnetic scattering techniques to examine structures on nanoscopic length scales. We are particularly interested in the way cellular activities are coordinated in space and time.

Biophysical methods and models

We develop new analytical methods to study biological structure and mechanistic cell behavior. These include physical methods based on fluorescence correlation spectroscopy (FCS), quantitative optical and atomic force (AFM) microscopy, and Fourier transform analysis. In particular, we devise techniques that permit us to examine the motion of particles within concentrated polymer solutions and dense, interconnected polymer matrices. One application involved examining the translocation of viruses through vaginal secretions, with the goal of understanding 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 studies of individual virions indicate that most virus particles slow down more than 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 polymer network of the mucus following mechanical perturbation of a sample, giving rise to a combination of anomalous diffusion and occasional jumps that depend on the microenvironments of individual virus particles (see reference 1).

Also, we used AFM to obtain time-sequence images of individual, wet (unfixed) triskelia resting on mica surfaces that demonstrate conformational fluctuations of the individual triskelial legs. Other AFM studies of dried samples yielded images with resolution comparable to that obtainable by electron microscopy (see reference 2). We observed increased numbers of triskelion dimers and assembly intermediates, as well as structures with dimensions similar to those of clathrin cages, when triskelia were immersed in a low salt, low pH buffer. We are now applying the knowledge gained from these and related studies to investigate the mechanical properties of reconstituted clathrin cages, focusing on discovering how the binding of clathrin to accessory proteins such as AP-2 and AP-180 influences clathrin coat rigidity.

The above phenomena occur in relatively dense media, in which 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 may be distorted by multiple scattering. We therefore 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 along the axial plane. Also, the incident intensity at the focal plane declines. Depending on the scattering properties of the medium, one may need to account for such effects when using FCS to investigate optically dense tissue. We have begun to examine the effects of crowding by using FCS to study the positively charged protein RNase A as it moves through polymeric solutions of dextrans of various charges. Our ultimate goal is to mimic the diffusion of biomacromolecules in particular models of cell cytoplasm. Varying the salt concentration permits us to address molecular crowding and charge-mediated binding concurrently in a controlled manner.

Clathrin lattice formation and supramolecular assembly

Receptor-mediated endocytosis involves the formation of membrane vesicles surrounded by closed polyhedral, cage-like structures assembled from a three-legged heteropolymer composed of three clathrin heavy/light chain complexes joined at a common hub (the "clathrin triskelion"). In devising physical theories to understand how such clathrin-coated vesicles (CCVs) arise, we developed various new quantitative methods of physical analysis. For example, 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, but they exhibit a somewhat different conformation than when incorporated into a reconstituted clathrin basket, suggesting that the mechanical properties of triskelia must be taken into account. Therefore, we developed a novel scheme, based on SANS, to assess the flexibility of the triskelia, permitting us to determine quantities that previously could be deduced only by more indirect methodology. Results substantiate our earlier inferences that triskelia are semiflexible polymeric structures capable of bending when integrated into polyhedral coats of various sizes and shapes.

In a related study, undertaken to gain deeper knowledge of the effects of molecular crowders on supramolecular assembly, we employed a biomimetic model consisting of macroscopic rods immersed in a 'fluid' of small spheres, in which mechanical shaking played a role analogous to thermal excitation. We found that, depending on the number densities of the constituents, the rods self-assemble into linear polymer-like structures when confined to quasi two-dimensional spaces. These structures are also predicted by equilibrium simulations, suggesting that entropy maximization is the driving force for bundling (see reference 3). Such results may provide insight into the role of extraneous solution constituents in the assembly of oligomeric structures. This work has been extended to a general study of nematic order occurring in small systems where the proximity of physical boundaries causes alignment of the 'molecular' constituents (Phys Rev Lett, in press).

Complex systems biophysics

We continue to use advanced physical and mathematical methods to understand the biophysics of complex cellular activities. The role of phosphoinositide metabolism in the biogenesis of vesicles involved in cellular transport events is such a process. We recently formulated a mathematical model to investigate the involvement of 3′ phosphoinositides in the biogenesis of clathrin-coated and other endocytic vesicles. This description of receptor-mediated endocytosis 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 at specific sites is triggered by the binding of ligands to receptors and subsequent recruitment of clathrin-associated proteins. As a start, we devised a mathematical model for the energy of formation of a clathrin-coated pit (CCP), in which the energy is expressed in terms of the size and curvature of the pit. The model contains three terms, viz., the energy needed to bend the plasma membrane, a line tension energy, and the energy stored in chemical bonds. We showed that, for reasonable parameter values, a plot of the free energy of a CCP with size shows an energy barrier that has to be crossed in order for a growing CCP to transform into a vesicle. We are also attempting to use a modified form of the model to rationalize the observed stochastic nature of cell response to the presence of a stimulus.

Tubulin polymers and cytoskeletal organization

The project focuses on the ability of small molecules to alter the cell cytoskeleton and, in particular, microtubules (MT). Small molecules that alter MT integrity and/or dynamics can affect intracellular trafficking and change the physical properties of the cytoplasm. We identified a number of new modified peptides derived from the natural microtubule destabilizer tubulysin. The new peptides show a range of antimicrotubule activity in assays with purified proteins as well as in cells. We also designed, synthesized, and tested a new, potent microtubule stabilizer based on the natural compound epothilone. In addition, we examined a new "second generation" of microtubule stabilizers, focusing on the natural compound peloruside; study of its binding to tubulin by hydrogen-deuterium exchange mass spectrometric methods led to new insights into the mechansims of normal microtubule assembly. We also showed that an old chemotherapy agent, a nitrosourea, may affect microtubule stability indirectly by altering the activity of the protein stathmin, which results in reduced migration and invasion by malignant glioma cells. An intended application of the knowledge of MT-small molecule interactions is to identify novel drug therapies for parasite diseases by identifying small molecules that do not bind well to mammalian tubulin but do bind to parasite tubulin. The tubulin molecule is quite conserved evolutionarily, but differences do exist, and several molecules are known that can target, for example, yeast rather than mammalian tubulin or vice-versa (see reference 4). We are looking for molecules that will target Leishmania, which is the infectious agent that causes an important group of human diseases. We identified several small molecules that show promise as selective agents, binding preferentially to Leishmania tubulin over mammalian tubulin and preventing parasite multiplication inside human macrophage cells. To screen for these drugs, we developed methods to purify tubulin from these cells and also developed methods to quantitate drug binding to tubulin based on changes in sulfhydryl chemistry.

Natural products have historically been the source of most of the anti-mitotic small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds in this study. Some, such as the new microtubule-stabilizing compound peloruside, are natural products. Others, such as analogs of the microtubule-stabilizing compound epothilone, and analogs of the microtubule-destabilizing peptide tubulysin, are derived by synthesis based on the structure of known natural compounds. We also discovered anti-mitotic compounds from a high-throughput screen of libraries of synthetic molecules whose structures are unrelated to particular natural compounds. These compounds all exert their actions through binding to tubulin, the subunit protein of microtubules. We showed that some effects of microtubule-active drugs are cell-type specific. An example of this is our demonstration that exposure of neural cells to microtubule-depolymerizing drugs results in a rapid degradation of tubulin following the expected microtubule depolymerization (see reference 5).

Additional Funding

  • Joint NIST-NIH National Research Council Postdoctoral Fellowship to Dr. Ji Youn Lee


  • Boukari H, Brichacek B, Stratton P, Mahoney SF, Lifson JD, Margolis L, Nossal R. Movements of HIV-virions in human cervical mucus. Biomacromolecules. 2009 10:2482-2488.
  • Kotova S, Prasad K, Smith PD, Lafer EM, Nossal R, Jin AJ. AFM visualization of clathrin triskelia under fluid and in air. FEBS Lett. 2010 584:44-48.
  • Galanis J, Nossal R, Harries D. Depletion forces drive polymer-like self-assembly in vibrofluidized granular materials. Soft Matter. 2010 6:1026-1034.
  • Sackett DL, Sept D. Making drug design second nature. Nat Chem. 2009 1:596-597.
  • Huff LM, Sackett DL, Poruchynsky MS, Fojo T. Microtubule-disrupting chemotherapeutics result in enhanced proteasome-mediated degradation and disappearance of tubulin in neural cells. Cancer Res. 2010 70:5870-5879.


  • Sergey Bezrukov, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • Beda Brichacek, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • Robert A. Fecik, PhD, University of Minnesota, Minneapolis, MN
  • Tito Fojo, MD, Medical Oncology Branch, NCI, Bethesda, MD
  • Amir Gandjbakhche, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • Daniel Harries, PhD, Hebrew University, Jerusalem, Israel
  • Ferenc Horkay, PhD, Program on Physical Biology, 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 Health Science Center, San Antonio, TX
  • Leonid Margolis, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • John Park, MD, Surgical Neurology Branch, NINDS, Bethesda, MD
  • Kondury Prasad, PhD, University of Texas Health Science Center, San Antonio, TX
  • Jason Riley, PhD, University College London, London, UK
  • Tatiana Rostovtseva, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • Dave Schriemer, PhD, University of Calgary, Calgary, Canada
  • David Sept, PhD, University of Michigan, Ann Arbor, MI
  • Candida Silva, PhD, Program on Physical Biology, NICHD, Bethesda, MD
  • Al Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD


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