Previous work in this laboratory by scanning transmission electron microscopy (STEM) has shown that Tetrahymena 21S dynein consist of a bouquet of three globular heads connected by three separate strands to a common base. Mass analysis by integration of electron scattering intensities gave a molecular weight of 2x10 to the 6 g/mole and indicated that two of the globular heads were identical with a mass of approximately 400 kdaltons while the third head exhibited a mass of approximately 550 kdaltons. Moreover, the present data suggests that the rootlike base of the bouquet forms the structural attachment site and the globular heads interact with the B-subfiber in an ATP-dependent reaction coupled to force production. Specifically, the current proposal seeks to follow up on these studies to (1) establish the absolute orientation of the molecule with respect to the A-subfiber of the outer doublet. (2) Determine whether two heads are identical and one head is different. (3) Localize the ATP and microtubule binding sites. (4) Localize the major polypeptides of the dynein molecule. (5) Begin to address the question of the roles of the three dynein heads in motility. These problems will be addressed by STEM and conventional TEM analysis of isolated dynein, intact ciliary outer doublets and dynein subfragments produced by proteolytic digestion. Monoclonal antibodies will be used to localize functional domains of the dynein molecule. This structural analysis is absolutely essential to the longer term objectives of establishing the mechanism of force pro-production and control of the dynein in ciliary motility and for subsequent analysis of dynein-like ATPases in other microtubule-dependent movements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032023-03
Application #
3280577
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1983-04-01
Project End
1986-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
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