Structural and regulatory aspects of ciliary outer arm dynein
Barkalow, Kurt Lewis
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Ciliary 22S dynein comprises the outer dynein arm which plays an important role in force production driving ciliary motility. I have studied this molecule's structural characteristics, as well as mechanisms by which it is regulated. There are two principal forms of the 22S dynein arm: the bouquet, where the three globular domains are spatially separated, and the compact arm, where they are aligned and folded together. When sliding of an isolated axoneme is induced by ATP at least 57% of total attached arms on exposed doublets can be identified in the compact form, while only about 2% are bouquets. When axonemes with exposed doublets are treated with 0.5 M KCl, the compact arms and the dynein H-chains disappear from the doublets, while isolated bouquets and dynein H-chains appear in the medium, suggesting that, as they are solubilized, the compact arms give rise to the bouquets. Computer modelling indicates that the dimensions of the bouquet are incompatible with the interdoublet volume available for an arm in the axoneme. These results suggest that the compact arm, rather than the bouquet, is the functional form of the arm in the intact axoneme. This model of the dynein arm structure has important implications for the predicted mechanochemistry of dynein. A 29 kDa polypeptide (p29) is directly associated with 22S dynein and is likely to be a dynein light chain that modulates dynein activity as a function of its cAMP-dependent phosphorylation state. Studies using partially purified p29 have shown it to be associated with 22S dynein specifically by a tight ionic interaction with one of the three globular domains, probably the of heavy chain. In vitro microtubule translocation after recombination of 22S dynein with cAMP-phosphorylated 29 is 1.4x faster than controls. Furthermore, p29 from Paramecium can specifically bind to 22S dynein from Tetrahymena suggesting that p29, or its homolog, may function to regulate dynein microtubule interactions in other organisms.
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