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]
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