The regulation of expression and targeting of dynein heavy chains in Paramecium tetraurelia

The objective of the present study is to investigate the regulation of dynein heavy chain expression and how dynein is targeted to its proper location for functioning within the cell. Dynein is a motor protein that utilizes energy from ATP hydrolysis to translocate along microtubules. There are two functional classes of dynein. Axonemal dyneins provide the force for the bending of eukaryotic cilia and flagella; cytoplasmic dyneins are involved in vesicle movements and nuclear migration. The single-celled ciliate Paramecium expresses at least 12 different dynein heavy chain isoforms; we have cloned and completely sequenced two of these dynein heavy chain isoforms, DHC-6 and DHC-8. Western blotting showed that DHC-6 was the $\beta$ heavy chain of outer arm axonemal dynein and DHC-8 was a cytoplasmic dynein. This enabled us to study the regulation of expression of these two functionally distinct dyneins within the same cell. Northern analysis demonstrated that axonemal dynein DHC-6 shows an increase in mRNA steady state levels after deciliation and that cytoplasmic dynein DHC-8 does not show this increase; nuclear run-on assays showed that the increase in DHC-6 expression after deciliation is due, at least in part, to an increase in transcription. DHC-6 and DHC-8 are differentially targeted; DHC-6 is targeted to the cilia and DHC-8 functions within the cytoplasm. It is my hypothesis that the amino terminal portion of the dynein heavy chain tethers the molecular cargo and functions to target the dynein. To test this hypothesis, I have constructed two epitope-tagged dynein constructs encompassing the amino terminal third and the central catalytic portion of the $\beta$ dynein heavy chain. These constructs were microinjected into the macronucleus of Paramecium in order to achieve DNA-mediated transformation. Southern blots showed that transformants are obtained by microinjection and Northern blots showed that the truncated dynein transcripts were made. Analysis of the transformants by Western blotting and immunofluorescence microscopy will be used to determine if either of the truncated dyneins localizes to the cilia