A conserved CaM- and radial spoke–associated complex mediates regulation of flagellar dynein activity

Abstract

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine 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 immunoprecipitation 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 sequence similarity to AAT-1, a protein originally identified 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 antibodies 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.

Flagellar and ciliary beating: the proven and the possible

“…Nonetheless, the idea that the spokes are the regulators of the beat cycle presently holds favor with many researchers in the field. This is because of the accumulated evidence for spoke-mediated regulation of microtubule sliding (Wargo and Smith, 2003;Smith and Yang, 2004;Yang et al, 2006;Dymek and Smith, 2007). Smith and Sale produced convincing evidence that the spoke apparatus can modulate doublet sliding and therefore might mediate coordinated sliding, possibly through interaction with the CP (Smith and Sale, 1992).…”

“…In this way, control by the CP-spoke axis might act to limit or terminate the action of selected dyneins, shaping the beat and altering the waveform for the different modes of swimming that are observed in Chlamydomonas. This might explain the role of Ca 2+ -binding elements on the spoke shafts (Yang et al, 2001) in controlling dynein activity (Dymek and Smith, 2007), as Ca 2+ can alter the waveform of the Chlamydomonas beat and change the swimming pattern. The results obtained by Nakano and colleagues using sea urchin sperm show that Ca 2+ can alter which specific dyneins are inhibited by association with the CP-spoke system (Nakano et al, 2003), and fit nicely with the interpretation that the CP-linked regulation of sliding is an element of the Ca 2+ response pathway.…”

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

“…Such a scenario could also be envisaged with the identified flagellar SAM protein that is phosphorylated at five clustered sites. An interesting candidate is also FAP288, which has an EF hand and thus may be involved in Ca 2ϩ signaling, which is known to play an important role in signaling in cilia (4,57).…”

PF19 encodes the catalytic subunit of katanin, p60, and is required for assembly of the flagellar central apparatus in Chlamydomonas

“…Axonemes were isolated as described previously (Dymek and Smith, 2007). Axonemes (10 mg) or whole cells (1610 6 cells) were resolved by SDS-PAGE using a 7% polyacrylamide gel.…”