Abstract

The actin filament is a key element in the dynamic machinery of motile cells and in the cytoskeleton of non-motile cells. Different assemblies of actin filaments fulfill different roles, even within the same cell. These assemblies differ both in their function and in their filament organization. We propose experiments to discover the properties of actin which make it a common element in the cytoskeletal structures. We also plan to study how these cytoskeletal structures are constructed. The approach, using combined techniques of cryo and conventional electron microscopy and image analysis, is to study the structure of isolated actin filaments, free and in combination with other proteins, actin bundles, and a cytoplasmic actin gel. In particular, we plan: 1. a study of actin and its auxiliary proteins in motile bundles obtained from the sperm of the horseshoe crab. The filaments obtained from this bundle are well ordered and therefore suitable for image analysis. The resulting three-dimensional maps will reveal the organization of the component proteins and will provide a map of the core actin filament. We will use the latter, together with the existing map of the subunit derived by X-ray analysis, to determine a near atomic model of the actin filament. 2. a study of the structure composition and assembly of the cuticular plate, an in vivo actin gel. This will be the first naturally occurring actin gel whose structure will be known in some detail. 3. a study of the structure of the actin-villin bundles. Villin is an actin-bundling and filament-severing protein. Images of the bundle in ice may reveal the structure of villin, how it interacts with actin and how it is able to sever filaments. 4. an in situ study of the actin cytoskeleton in yeast. Only three proteins and seven genes appear to be involved in the actin cytoskeleton. Because of its simplicity and genetic characterization, this system may be the ideal one in which to combine genetics, biochemistry and structural studies. The point of the proposed work is to see how tractable the system is for structural studies.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026357-14
Application #
3273859
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1979-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
14
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
Brandeis University
Department
Type
Schools of Arts and Sciences
DUNS #
616845814
City
Waltham
State
MA
Country
United States
Zip Code
02454

  Publications

Mercogliano, Christopher P; DeRosier, David J (2007) Concatenated metallothionein as a clonable gold label for electron microscopy. J Struct Biol 160:70-82
Mercogliano, Christopher P; DeRosier, David J (2006) Gold nanocluster formation using metallothionein: mass spectrometry and electron microscopy. J Mol Biol 355:211-23
Volkmann, Niels; Liu, HongJun; Hazelwood, Larnele et al. (2005) The structural basis of myosin V processive movement as revealed by electron cryomicroscopy. Mol Cell 19:595-605
Tilney, Lewis G; DeRosier, David J (2005) How to make a curved Drosophila bristle using straight actin bundles. Proc Natl Acad Sci U S A 102:18785-92
Tilney, Lewis G; Connelly, Patricia S; Ruggiero, Linda et al. (2004) The role actin filaments play in providing the characteristic curved form of Drosophila bristles. Mol Biol Cell 15:5481-91
Sukow, Catherine; DeRosier, David J (2003) Order, disorder, and perturbations in actin-aldolase rafts. Biophys J 85:525-36
Volkmann, Niels; Ouyang, Greta; Trybus, Kathleen M et al. (2003) Myosin isoforms show unique conformations in the actin-bound state. Proc Natl Acad Sci U S A 100:3227-32
Li, Huilin; DeRosier, David J; Nicholson, William V et al. (2002) Microtubule structure at 8 A resolution. Structure 10:1317-28
Volkmann, N; DeRosier, D; Matsudaira, P et al. (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
DeRosier, D J; Tilney, L G (2000) F-actin bundles are derivatives of microvilli: What does this tell us about how bundles might form? J Cell Biol 148:6-Jan

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