X-Ray Fiber Diffraction Recordings from Oriented Demembranated Chlamydomonas Flagellar Axonemes

Abstract

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the α-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

X‐ray fiber diffraction analysis shows dynamic changes in axial tubulin repeats in native microtubules depending on paclitaxel content, temperature and GTP‐hydrolysis

“…Recently, we developed a new technique for aligning flagellar axonemes in shear‐flows to observe oriented X‐ray fiber diffraction [Oiwa et al, ; Sugiyama et al, ; Toba et al, ]. Because we could align filamentous specimens in seconds and with angular deviations of <5°, we applied this technique to the X‐ray fiber diffraction analysis to identify dynamic structural changes or conversions of native microtubules.…”

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists

“…Chlamydomonas reinhardtii is a biflagellar single‐cell green algae having dynein motors at its outer and inner arms of the flagella. In C. reinhardtii , a cooperative action of multiple dynein motors triggered by ATP produces propulsive force for the beating motion of its flagella [32–34] . We used a uniflagellar mutants of C. reinhardtii , which is known to undergo rotational motion [35–36] .…”

“…In C. reinhardtii, a cooperative action of multiple dynein motors triggered by ATP produces propulsive force for the beating motion of its flagella. [32][33][34] We used a uniflagellar mutants of C. reinhardtii, which is known to undergo rotational motion. [35][36] To study the effects of ATP antagonists on the behavior of C. reinhardtii, the demembranated model was prepared according to the previous studies.…”

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules

“…The axonemal components are arranged on DMTs as axial repeats that are multiples of the 8-nm tubulin-dimer repeat. Major axonemal components, such as the dynein arms and radial spokes, have 96-nm structural repeats that represent the fundamental functional unit (Bui et al, 2008;Toba et al, 2015). Previous studies have shown that MIPs possess such structural repeats: MIP1a and MIP1b make an array alternatingly and possess repeats with 16-nm spacing; those of MIP2a exhibit 16-and 32-nm spacing; those of MIP2b have 48-nm spacing; those of MIP3a and MIP3b have 16-nm spacing; and those of MIP4 exhibit 48-nm spacing.…”