Identification and characterization of DHC1b, a novel cytoplasmic dynein
Abstract
Dynein is a large, microtubule dependent motor enzyme which transduces the energy of ATP hydrolysis into movement. There are two classes of dynein motors, axonemal and cytoplasmic dyneins. At least 12 axonemal dynein isoforms participate in the bending of cilia and eukaryotic flagella. A single cytoplasmic dynein heavy chain, DHC1a, is implicated in retrograde transport of membranous organelles, spindle assembly and movement of kinetochores during mitosis, nuclear migration and axonal transport. Recent studies suggest that some species express more than one cytoplasmic dynein isoform. DHC1b has been recently identified in sea urchin, rat, C. elegans and Tetrahymena. These studies describe the isolation and characterization of DHC1b from rat. DHC1b is present in all tissues examined and is distinguished from DHC1a. The DHC1b mRNA expression increases with ciliogenesis, while that of DHC1a does not. However, DHC1b is present exclusively in the cytoplasm and is excluded from the cilia. DHC1a and DHC1b proteins can be separated by sucrose density centrifugation; 1b sediments as a monomer while 1a sediments as a dimer. Polyacrylamide gel electrophoresis of testis cytoplasmic dynein shows that the apparent molecular mass of 1b is approximately 60,000 Daltons smaller than 1a. The specific ATPase activity of DHC1b is about five fold higher than DHC1a, but DHC1b is stimulated by microtubules to a lesser extent than is DHC1a. DHC1a undergoes photolysis in the presence of vanadate and UV light, producing two fragments. DHC1b undergoes photolysis under the same conditions, but cleaves into 2-3 pairs of fragments, suggesting that there may be as many as three different DHC1b isoforms. Peptide specific antibodies were produced which differeniate DHC1b from DHC1a. Immunofluorescent staining of ciliated rat tracheal cells with the peptide-specific antibodies shows that DHC1b accumulates to the apical region of epithelial cells, while DHC1a is more diffusely spread throughout the cytoplasm. Neither of the cytoplasmic antibodies stain the cilia, but anti-axonemal antibodies exclusively stain the cilia. The data are consistent with the hypothesis that DHC1b is a cytoplasmic dynein that functions in production or maintenance of axonemes in some tissues.
Degree
Ph.D.
Advisors
Asai, Purdue University.
Subject Area
Cellular biology|Anatomy & physiology|Animals
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