Dissection of cyclic nucleotide signaling pathways in flagellar axonemes of Chlamydomonas

Jessica Belen-Rivera, Ashley Solmonson, Vincent Maresca, Terah McClendon, Joshua Farthing, Victoria Alfaro, and Anne Gaillard

Sam Houston State University, Department of Biological Sciences, Huntsville TX 77341, USA

Numerous physiological and pharmacological studies have shown that cAMP is a regulator of axonemal motility in Chlamydomonas flagellar axonemes, and that cAMP signaling involves both the central pair/radial spoke control system and I1 dynein (f dynein). In order to further characterize this signaling pathway, a genetic cross between an I1 dynein mutant (ida1) and a mutant defective in the PKA-binding domain of RSP3 (388) was performed. Progeny containing both mutations exhibit a percentage of motility similar to ida1 cells, while swimming speed is intermediate of ida1 and 388. Also, like both ida1 and 388, ida1/388 double mutants display reduced phototaxis. This study also examined the role of cGMP in control of axonemal motility. Pharmacological studies revealed that inhibitors of cGMP-dependent protein kinase (PKG) reduce motility of wild-type cells. Based on Western blots using a peptide antibody to flagellar PKG, PKG is present in the axoneme, but is not localized exclusively to the dynein arms, radial spokes, central pair, or dynein regulatory complex. Furthermore, sucrose fractionation and immunoprecipitation studies revealed that PKG is part of an axonemal complex of proteins, extractable with 0.6 M NaCl.

e-mail address of presenting author: argaillard@shsu.edu