Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella

Abstract

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa

“…4B). Similar to localization of TCTE1-FLAG along the flagellum, the interaction studies are also in agreement with previous reports on TCTE1 (23,32).…”

“…S6). This observation is consistent with work in Chlamydomonas Drc5 mutants showing little to no observable defects in axoneme organization (23,33). The intraflagellar transport (IFT) machinery is required for assembling the flagellum during spermiogenesis (44,45).…”

“…This complex interconnects the A tubule with the adjacent microtubule pair's B to prevent sliding between tubules from the forces generated by axonemal dynein (35). Several mutants in the nexin complex disrupt the coordinated movement of the axoneme, leading to irregular flagella beating (7,23,32,36,37).…”

“…Drc5 is the Tcte1 ortholog in these unicellular green algae. Deletion and proteomic analyses in Chlamydomonas identified DRC5 as a component of the N-DRC (23,32,33). Nexin or "bridge" was the name for a structure observed between the outer doublets of the 9+2 arrangement from electron micrographs (32,34).…”

“…4A) (41)(42)(43). Based on cryo-electron tomography of Chlamydomonas wild-type and mutant axonemes (23,32), DRC5 (TCTE1), -3, -6, and -7 have been mapped proximal to each other within the DRC complex (Fig. 4B).…”