Two Anti-radial Spoke Monoclonal Antibodies Inhibit Chlamydomonas Axonemal Motility by Different Mechanisms

Daniel White,Soroush Aghigh,Ilana Magder,Jacky Cosson,Philippe Huitorel,Claude Gagnon

doi:10.1074/jbc.m414114200

Daniel White, Soroush Aghigh + Show 4 more

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https://doi.org/10.1074/jbc.m414114200

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Abstract

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.

Highlights

In the 9 ؉ 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules
When L2H12 or L3G4 monoclonal antibodies (mAb) was added into the assay mixture, the time needed to inhibit the motility and bring the percentage of motile cells to 10% or below varied according to the concentration and mAb used (Fig. 1)
The mAb generated (L2H12 and L3G4) against two individual RSP, each one found to be localized on a different region of the radial spoke, showed potent inhibitory action on the motility of demembranated-reactivated Chlamydomonas cells (Fig. 1)

Summary

Introduction

In the 9 ؉ 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Further insight into the radial spoke function has been provided by studies on suppressor mutations restoring motility in radial spoke mutants without restoration of these structures [13,14,15] Their results suggested that radial spokes are not essential for bending to occur but, rather, contribute in the modulation of the microtubule doublets, sliding to give rise to the asymmetric flagellar movement typical of wild-type Chlamydomonas. All these findings suggest a role for the radial spokes in the regulation of flagellar movement likely via a complex signal transduction system [21,22,23,24], but the precise function of radial spoke is still unclear

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