|SSRL Science Highlight - December 2003|
Olivier Pelletier, Elena Pokidysheva, Linda S. Hirst, Nate Bouxsein, Youli Li
and Cyrus R. Safinya
Materials and Physics Departments, Biomolecular Science & Engineering Program, Materials Research Laboratory, University of California, Santa Barbara, California 93106
With the emerging proteomics era, the biosciences community is now challenged to elucidate the structures and functions of a large number of interacting proteins. Our understanding of modern biology has to a significant degree resulted from our knowledge of the structures of biological molecules, which provide direct information relating to function. The elucidation of in vitro supramolecular structures —spanning length scales from nanometers to microns— designed to model in vivo conditions of interacting proteins required for cell function, will require interdisciplinary approaches and techniques. An important goal in biophysics is the understanding of interactions leading to supramolecular structures of cytoskeletal proteins and associated biomolecules, and most important, the elucidation of the roles the structures play in cell functions.
Our group at the University of California at Santa Barbara (UCSB) has recently reported on the structure of filamentous (F) actin complexed with the actin cross-linking protein a-actinin (1). On the nanometer scale, synchrotron x-ray scattering and diffraction carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), has revealed a structural transition from a loose network of filaments at low cross-linker concentrations, to a disordered quasi-square lattice of actin fibers within bundles, at cross-linker concentrations near physiological concentrations. On the micron scale, laser scanning confocal microscopy has revealed a relatively rigid, frequently branching, three-dimensional network of bundles with characteristic mesh size of the order of the persistence length of F-actin.
The cell cytoskeleton comprises three negative charged filamentous proteins, which include, filamentous-actin with a 8.5 nm diameter, the intermediate filaments, 10 nm in diameter, and microtubules, 25 nm in diameter. The cytoskeleton is involved in a range of cell functions including mechanical stability, cell locomotion, intracellular trafficking and signal transduction. We will describe recent work on interactions and structures in supramolecular assemblies of the actin cytoskeleton under in vitro conditions.
We show in Fig. 1 (left) laser scanning confocal microscope images of the actin cytoskeleton in mouse fibroblast cells. (The image is showing only the section closest to the cover slip to which the cell is attached.) The actin cytoskeleton provides a structural framework for the mechanical stability of eukaryotic cells and is involved in functions including cell adhesion, motility, and division. Actin is found both in a monomeric globular (G) actin state and as polymerized filamentous (F) actin. Bundles, comprised of closely packed parallel arrangements of F-actin, and networks, containing F-actin crisscrossed at some large angle, form the most common known supramolecular structures in cells. Interactions between F-actin and distinct actin cross-linking proteins, may lead to two-dimensional (2D) networks and bundles of F-actin interacting with the plasma membrane to determine cell shape, or 3D networks of F-actin imparting gel-like properties to the cytosol.
In Figure (Fig. 1, left, broken arrow), actin bundles can be seen traversing the length of a cell near the center of the image. These bundles are components of stress fibers, which end in focal adhesion spots responsible for cell adhesion, for example, to the extracellular matrix in the space between cells in vivo. Radial bundles of lamellipodium in membrane-protrusions (Fig. 1, left, solid arrow) are also associated with adhesion complexes.
When filamentous actin is allowed to mix in-vitro with a-actinin, an actin
cross-linking protein purified from cells, a remarkable new type of
biologically inspired polymer-network is spontaneously formed with
fundamentally new properties. Three-dimensional laser scanning confocal
microscopy carried out in our laboratory at UCSB has revealed a network of
bundles on the micron scale (Fig. 1, right). This is in contrast to the
commonly observed network of single filaments observed in cells. The branching
(bifurcation) of the bundles on this meso-scale is evident and leads to a
well-defined mesh size (1).
A separate motivation for the work described here was provided by our NIH funded research project within the group focused on developing optimal non-viral cationic lipid (CL) carriers of genes for gene delivery. Gene carriers based on lipids — rather than on engineered viruses — are among the most promising technologies for delivering genes into cells for gene therapy and therapeutics (2,5). Indeed, nearly one quarter of ongoing gene therapy clinical trials worldwide are conducted with non-viral methods including lipids, polymers, and naked DNA (http://www.wiley.co.uk/genetherapy/clinical/). Synchrotron x-ray diffraction work at SSRL by our group has solved the structures formed by CL-DNA complexes (6,7).
From a fundamental perspective, a full understanding of gene transfer technology by cationic lipids will emerge once the interactions between exogenous lipid and the biomolecules of the cell are understood. Work by our group at SSRL, which studied the interactions between CLs and F-actin in-vitro, showed that CL-actin complexes may spontaneously assemble into multilamellar tubules (8). The study reported here constitutes an important step at elucidating the structure of F-actin/a-actinin complexes, which will be followed by future experiments probing the interactions between F-actin/a-actinin and CL-DNA complexes mimicking gene delivery conditions. Thus, one may expect that actin filaments may dissociate from bundles in the cell cytoplasm and interact with and remove cationic lipids from CL-DNA complexes, thus, facilitating DNA release from complexes in cells for expression of genes.
Acknowledgements The work described in this report was supported by the National Institutes of Health grant GM-59288, the National Science Foundation, grants DMR-0203755 and CTS- 0103516, and the Department of Energy's Office of Basic Energy Sciences under contract number W-7405-ENG-36 with the University of California. X-ray diffraction was conducted at The Stanford Synchrotron Radiation Laboratory supported by the US Department of Energy.
SSRL Highlights Archive
|Last Updated:||10 DEC 2003|
|Content Owner:||Cyrus Safinya|
|Page Editor:||Lisa Dunn|