Regulation of Paramecium ciliary activity by thecAMP-stimulated phosphorylation of a 29 kD dynein light chain

Abstract

This thesis demonstrates a mechanism by which ciliary dynein mechanochemistry may be regulated in Paramecium. Ca{dollar}\sp{lcub}2+{rcub}{dollar} and cyclic nucleotides are second messengers for the control of ciliary activity. Computerized analysis of Mg{dollar}\sp{lcub}2+{rcub}{dollar}-ATP reactivated Triton X-100 permeabilized cells shows that the permeabilized cells respond to experimentally added second messengers: they swim backward in {dollar}>{dollar}10{dollar}\sp{lcub}-6{rcub}{dollar}M Ca{dollar}\sp{lcub}2+{rcub}{dollar} or swim forward faster in {dollar}>{dollar}10{dollar}\mu{dollar}M cAMP. Ciliary beat form of the forward swimming reactivated cells is virtually indistinguishable from that of intact cells. Phosphorylation of isolated, demembranated axonemes was studied in vitro using {dollar}\delta{dollar}-{dollar}\sp{lcub}32{rcub}{dollar}P-ATP or {dollar}\delta{dollar}-{dollar}\sp{lcub}35{rcub}{dollar}S-ATP to uncover proteins which alter their phosphorylation level in response to (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) or (cAMP). A 29 kD polypeptide was found to be phosphorylated or thiophosphorylated in the presence of micromolar cAMP, and Ca{dollar}\sp{lcub}2+{rcub}{dollar} at pCa 4 inhibits this phosphorylation. This polypeptide, either phosphorylated or thiophosphorylated in the presence of cAMP, copurifies with 22S dynein by two different procedures: a microtubule-affinity method and sucrose density gradient centrifugation (cAMP 22S dynein). The thiophosphorylated polypeptide was not detected in 22S dynein prepared from axonemes thiophosphorylated in the absence of cAMP (control 22S dynein) or in the presence of cAMP at pCa 4 (cAMP + Ca{dollar}\sp{lcub}2+{rcub}{dollar} 22S dynein). In vitro microtubule translocation assays were used to test the functional aspects of phosphorylation of the 29kD polypeptide in 22S dynein. cAMP 22S dynein caused significantly (40%) faster in vitro microtubule translocation compared to control or cAMP + Ca{dollar}\sp{lcub}2+{rcub}{dollar} 22S dynein, which velocities were nearly identical. Thus the 29kD polypeptide acts as a dynein regulatory light chain which controls the speed of microtubule translocation by a cAMP-stimulated, Ca{dollar}\sp{lcub}2+{rcub}{dollar}-sensitive phosphorylation. The 29kD polypeptide does not copurify with 14S dynein. To test whether activation of the 22S or 14S dynein can account for the activation of ciliary activity, Triton-permeabilized cells were preincubated with ATP-{dollar}\delta{dollar}-S in three different conditions: absence of cAMP (control cells), presence of cAMP (cAMP cells) and presence of cAMP at pCa 4 (cAMP + Ca{dollar}\sp{lcub}2+{rcub}{dollar} cells)--and then reactivated under identical conditions. Motion analysis revealed that cAMP cells showed significantly higher forward swimming velocity than control or cAMP + Ca{dollar}\sp{lcub}2+{rcub}{dollar} cells, both of which had the same mean velocity. Phosphorylation of the 29kD polypeptide not only regulates isolated 22S dynein but also can account for a major regulatory mechanism that controls ciliary motility in vivo.