Actin and cell shape and motility:

Actin is best known as the railroad tracks along which the myosin motors pull themselves. Actin in its filamentous form is a helical arrangement of subunits upon which myosin steps as it walks its way along the filament. But actin lies at the heart of other kinds of cellular machines such as the hair cell of the inner ear. Hair cells transduce sound-generated motion into electrical impulses, which is how we hear. Protruding from each hair cell is an organ pipe array of rigid cellular protrusions called stereocilia. Fluid motion, driven by sound, moves the stereocilia, which by regulating ion channels, modulate the electrical potential across the hair cell's membrane. Thus by this path, the stereocilia convert sound to electrical changes.

The core of every stereocilium is an actin bundle. The stereocilium is held erect because its actin bundle sends rootlet into the body of the cell where it is anchored in an actin gel. Fimbrin, an actin-bundling protein, crosslinks the filaments providing integrity and rigidity to the bundle. Because the arrangement of filaments in bundles is not crystalline, a condition which allows study by conventional methods, we divided the structure into subcomplexes to make the problem tractable. We can better study the parts individually and then reassemble them into the bundle. This is the divide and conquer approach, which we have undertaken with our collaborators Prof. Paul Matsudaira (The Whitehead Institute, M.I.T.) and Steve Almo (Albert Einstein College of Medicine).

Electron density map of F-actin (solid blue) overlaid with the atomic model of F-actin (white model) (Hanein et al, JCB 139 pp. 387-396 (1997)). Click here for details

Fimbrin, a protein containing two actin binding domains, is a member of a superfamily of actin binding proteins known as the calponin homology superfamily. All members of this superfamily appear to possess homologous actin binding domains. The atomic structure of one of the fimbrin actin-binding domains is known from x-ray crystallographic studies. Similarly, the actin subunit is known to atomic resolution. Unfortunately, no one has been able to produce a crystal containing actin and fimbrin for study by x-ray crystallography. To get an atomic model of an actin-fimbrin interaction, we used electron microscopy and digital image processing to produce a three dimensional map of a complex of actin and the known actin binding domain of fimbrin. With a resolution of about 2 nanometers, the map allowed us to visualize single molecules and even the domains that comprise them. We then docked the atomic models of the subunits into our actin-fimbrin map to generate an atomic model of the complex.

Our next step was to find a suitable subassembly of the bundle so that we might visualize the whole fimbrin molecule in its role as a crosslinking protein. The actin-fimbrin raft is a two-dimensional array of actin filaments crosslinked by fimbrin molecules. The idea is that a raft corresponds to one row of filaments in a three dimensional bundle. We make the rafts by aligning filaments on a lipid sheet and crosslinking them with fimbrin. We are beginning the analysis of rafts by electron microscopy and image analysis.

Click here for files associated with the paper "An atomic model of fimbrin binding to F-actin and it's implications for filament crosslinking and regulation" by D. Hanein, N. Volkmann, S. Goldsmith, A-M. Michon, W. Lehman, R. Craig, D. DeRosier, S. Almo, and P. Matsudaira in Nature Structural Biology 5:787-792 (1998).

Bacterial propulsion:

The bacterial flagellum, which propels many types of bacteria, has a long corkscrew-shaped propeller attached to a rotary motor by a drive shaft and short flexible universal joint. The drive shaft passes through a bushing that holds the motor firm in the cell's envelope. The motor, which is powered by the proton gradient across the cell's membrane, can spin at 60,000 rpm. The tiny machine is made of thousands of protein molecules but requires only 40 genes. Using the electron microscope and image analysis, we were able to generate three-dimensional images of the flagellum. In our images we can visualize the arrangements and structures of the component protein parts. The subunits of the propeller, universal joint and drive shaft wind around in a continuous helical filament. The motor's subunits form rings rather than helices. Of the 40 proteins, only 5 appear to be responsible for generating torque and reversing the direction of the motor's rotation. We are identifying and determining the locations of these proteins in our images.

Selected publications

Morgan, D.G., Owen, C., Melanson, L.A., DeRosier, D.J. 1995. Structure of bacterial flagellar filaments at 11Å resolution: packing of the a-helices. J. Mol. Biol. 249: 88-110. [abstract]

Owen, C.H., Morgan, D.G. and DeRosier, D.J. 1996. Image analysis of helical objects: The Brandeis helical package. J. Struct. Biol. 116:167-175. [abstract]

Kihara, M., Francis, N.R., DeRosier, D.J. and Macnab, R.M. 1996. Analysis of a FliM-FliN flagellar switch fusion mutant of Salmonella typhimurium. J. Bacteriol. 178:4582-4589. [abstract]

Hanein, D., Matsudaira, P., and DeRosier, D.J. 1997. Evidence for a conformational change in actin induced by fimbrin (N375) binding. J. Cell. Biol. 139:387-396. [abstract] [full text]

Thomas, D.R., Morgan, D.G., and DeRosier, D.J. 1999. The rotational symmetry of the C ring and a model for the mechanism of the bacterial flagellar rotary motor. Proc Natl Acad Sci 96:10134-10139. [abstract] [full text]

Hanein, D. and DeRosier, D.J. 1999. A new algorithm to align three-dimensional maps of helical structures. Ultramicroscopy 76:233-238. [abstract]

DeRosier, D., Stokes, D.L. and Darst, S. 1999. Averaging data derived from images of helical structures with different symmetries. J. Mol. Biol. 289:159-65. Erratum. J. Mol. Biol. 289:1145-50. [abstract]

DeRosier, D.J. 2000. Correction of high resolution data for curvature of the Ewald sphere. Ultramicrosc. 81:83-98.

DeRosier, D.J. and Tilney, L.G. 2000. F-actin bundles are derivatives of microvilli: what does this tell us about how bundles might form? J Cell Biol. 2000 Jan 10;148(1):1-6. [abstract] [full text]

Yonekura K, Maki S, Morgan DG, DeRosier DJ, Vonderviszt F, Imada K, Namba K. (2000) The bacterial flagellar cap as the rotary promoter of flagellin self-assembly. Science. 290:2148-52. [abstract]

Volkmann N, Hanein D, Ouyang G, Trybus KM, DeRosier DJ, Lowey S. (2000) Evidence for cleft closure in actomyosin upon ADP release. Nat Struct Biol. 7:1147-55. [abstract]

Volkmann, N., DeRosier, D., Matsudaira, P., Hanein, D. (2001) An atomic model of actin filaments cross-linked by fimbrin and its implications for bundle assembly and function. J Cell Biol 153: 947-56. [abstract]

Thomas D, Morgan DG, DeRosier DJ. (2001) Structures of bacterial flagellar motors from two FliF-FliG gene fusion mutants. J Bacteriol. 183:6404-12. [abstract]

Francis NR, Levit MN, Shaikh TR, Melanson LA, Stock JB, DeRosier DJ. (2002) Subunit organization in a soluble complex of Tar, CheW, and CheA by electron microscopy. J Biol Chem. 2002 Jul 15.

Sukow C, DeRosier DJ. (2003). Order, disorder, and perturbations in actin-aldolase rafts. Biophys J. 85(1):525-36.

Volkmann N, Ouyang G, Trybus KM, DeRosier DJ, Lowey S, Hanein D. (2003) Myosin isoforms show unique conformations in the actin-bound state. Proc Natl Acad Sci U S A. 100:3227-32. [abstract]

Young HS, Dang H, Lai Y, DeRosier DJ, Khan S. (2003) Variable symmetry in Salmonella typhimurium flagellar motors. Biophys J. 84:571-7. [abstract]

Francis NR, Wolanin PM, Stock JB, DeRosier DJ, Thomas DR. (2004) Three-dimensional structure and organization of a receptor/signaling complex. Proc Natl Acad Sci U S A. 101:17480-5. [abstract]

Samatey FA, Matsunami H, Imada K, Nagashima S, Shaikh TR, Thomas DR, Chen JZ, DeRosier DJ, Kitao A, Namba K. (2004) Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism. Nature. 431:1062-8. [abstract]

Tilney LG, Connelly PS, Ruggiero L, Vranich KA, Guild GM, DeRosier D. (2004) The role actin filaments play in providing the characteristic curved form of Drosophila bristles. Mol Biol Cell. 15:5481-91. [abstract]

Shaikh TR, Thomas DR, Chen JZ, Samatey FA, Matsunami H, Imada K, Namba K, and DeRosier DJ. (2005) A partial atomic structure for the flagellar hook of Salmonella typhimurium. Proc Natl Acad Sci U S A, 102: 1023-1028. [abstract]

 

---

Last review: July 20, 2005