JCB: ARTICLE A conser ved CaM- and radial spoke–associated complex mediates regulation of ﬂagellar dynein activity Y G O L O I B L L E C F O L A N R U O J E H T Erin E. Dymek and Elizabeth F. Smith Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 F second messengers calcium and cyclic adenosine or virtually all cilia and eukaryotic ﬂagella, the monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immuno- precipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes ﬂagellar- associated protein 91 (FAP91), which shares considerable Introduction For eukaryotic cells that use cilia and flagella for motile func- tions, motility is commonly modulated in response to extracel- lular cues; this modulation may include changes in waveform (Brokaw et al., 1974; Brokaw, 1979; Bessen et al., 1980; Kamiya and Witman, 1984), beat frequency (Verdugo, 1980), or direc- tion of the effective stroke (Naitoh and Kaneko, 1972; Izumi and Miki-Noumura, 1985). Despite the diversity of responses between cell types, changes in motility are often preceded and mediated by changes in the intraflagellar concentrations of the second messengers calcium and cAMP. Ciliary and flagellar beating results from the spatial regulation of dynein activity along the axonemal microtubules (Satir, 1985). Our goal is to understand how changes in intraflagellar calcium concentra- tions are converted to changes in dynein-driven microtubule sliding to modulate motility. Substantial evidence from our laboratory and others indi- cates that the central apparatus and radial spokes form a signal transduction pathway that modulates ciliary and flagellar beat- ing in response to second messengers (for reviews see Porter and Correspondence to Elizabeth F. Smith: elizabeth.f.smith@dartmouth.edu Abbreviations used in this paper: AKAP, A-kinase anchor protein; CSC, CaM- and spoke-associated complex; DRC, dynein regulatory complex; EST, expressed sequence tag; FAP, ﬂagellar-associated protein; RSP, radial spoke protein. The online version of this article contains supplemental material. © The Rockefeller University Press $30.00 The Journal of Cell Biology, Vol. 179, No. 3, November 5, 2007 515–526 http://www.jcb.org/cgi/doi/10.1083/jcb.200703107 sequence similarity to AAT-1, a protein originally identi- ﬁed in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of anti- bodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms. Sale, 2000; Smith and Yang, 2004). Using both functional and structural approaches, our previous studies demonstrated that calcium control of motility involves the regulation of dynein- driven microtubule sliding and that CaM is a key axonemal calcium sensor (Smith, 2002a,b; Wargo and Smith, 2003; Wargo et al., 2004). Based on these results, we postulate that the calcium sensor regulates the activity of specific dynein sub- forms and/or dynein arms attached to specific subsets of dou- blet microtubules, thus modulating the size and shape of ciliary/ flagellar bends. Understanding how CaM might regulate dynein activity to modulate ciliary motility requires the localization of CaM within the axoneme as well as the identification of CaM binding partners. Yang et al. (2001) have reported that a fraction but not all of the axonemal CaM associates with the radial spokes in Chlamydomonas reinhardtii. We have identified and character- ized additional CaM-binding proteins in C. reinhardtii flagella using an immunoprecipitation approach. We developed anti- bodies against a peptide antigen unique to the C terminus of C. reinhardtii CaM and used these antibodies to precipitate CaM from extracted axonemal proteins. We previously reported that eight polypeptides precipitate with CaM and that these polypep- tides form at least two different protein complexes (Wargo et al., 2005). One complex is comprised of five polypeptides in addi- tion to CaM and is associated with the C1 microtubule of the JCB 515