Research Article

Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

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Science  08 Jan 2021:
Vol. 371, Issue 6525, eabd4914
DOI: 10.1126/science.abd4914

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Glycylation regulates axonemal dyneins

Physiological functions of the microtubule cytoskeleton are expected to be regulated by a variety of posttranslational tubulin modifications. For instance, tubulin glycylation is almost exclusively found in cilia and flagella, but its role in the function of these organelles remains unclear. Gadadhar et al. now demonstrate in mice that glycylation, although nonessential for the formation of cilia and flagella, coordinates the beat waveform of sperm flagella. This activity is a prerequisite for progressive sperm swimming and thus for male fertility. At the ultrastructural level, lack of glycylation perturbed the distribution of axonemal dynein conformations, which may explain the observed defects in flagellar beat.

Science, this issue p. eabd4914

Structured Abstract

INTRODUCTION

Microtubules are key components of the eukaryotic cytoskeleton. Although they are involved in a wide variety of functions, microtubules are structurally highly similar across most cell types and organisms. It was suggested that a “tubulin code,” formed by combinations of tubulin posttranslational modifications, adapts individual microtubules to specific functions within living cells. However, clear-cut functional and mechanistic data verifying this concept are still scarce. Glycylation is among the least explored posttranslational modifications of tubulin and has, so far, exclusively been found on microtubules of cilia and flagella from a variety of species. Previous work has suggested that glycylation might be essential for cilia and flagella, but mechanistic insight remains lacking.

RATIONALE

Two enzymes from the tubulin-tyrosine ligase-like (TTLL) family, TTLL3 and TTLL8, are essential to initiate glycylation of tubulin in mammals. To entirely abolish glycylation at the organism level and to determine its physiological function, we generated a double-knockout mouse lacking both glycylating enzymes (Ttll3−/−Ttll8−/−). Inactivation of these two enzymes led to a lack of glycylation in all analyzed cilia and flagella. This allowed us to investigate the role of glycylation in the function of these organelles.

RESULTS

Despite the absence of glycylation in Ttll3−/−Ttll8−/− mice, no gross defects were observed at the organism and tissue levels. Motile ependymal cilia in brain ventricles as well as motile cilia in the respiratory tract were present and appeared normal. Sperm flagella were also assembled normally, and sperm were able to swim. However, in vitro fertility assays showed that male Ttll3−/−Ttll8−/− mice were subfertile. Computer-assisted sperm analyses revealed motility defects of Ttll3−/−Ttll8−/− sperm. Further analyses showed that lack of glycylation leads to perturbed flagellar beat patterns, causing Ttll3−/−Ttll8−/− sperm to swim predominantly along circular paths. This is highly unusual for mammalian sperm and interferes with their ability to reach the oocyte for fertilization. To determine the molecular mechanisms underlying this aberrant flagellar beat, we used cryo–electron tomography. The three-dimensional structure of the 96-nm repeat of the Ttll3−/−Ttll8−/− sperm axoneme showed no aberrations in its overall assembly. By contrast, the structure of both outer and inner dynein arms (ODAs and IDAs) was perturbed in Ttll3−/−Ttll8−/− flagella. Classification analysis showed that the incidence and distribution of pre-powerstroke and post-powerstroke conformations of ODAs and IDAs were altered in Ttll3−/−Ttll8−/− sperm. These ultrastructural findings indicate that glycylation is required to efficiently control the dynein powerstroke cycle, which is essential for the generation of a physiological flagellar beat.

CONCLUSION

Our work shows that tubulin glycylation regulates the beat of mammalian flagella by modulating axonemal dynein motor activity. Lack of glycylation leads to perturbed sperm motility and male subfertility in mice. Considering that human sperm are more susceptible than mouse sperm to deficiencies in sperm motility, our findings imply that a perturbation of tubulin glycylation could underlie some forms of male infertility in humans.

Tubulin glycylation controls sperm motility.

(A) Microtubules in sperm flagella are rich in tubulin posttranslational modifications. Mice deficient for the glycylating enzymes TTLL3 and TTLL8 lack glycylation. (B) Mammalian sperm swim in linear paths. In the absence of glycylation, abnormal, mostly circular swimming patterns are observed, which impede progressive swimming. (C) Absence of glycylation leads to perturbed distribution of axonemal dynein conformations in Ttll3−/−Ttll8−/− flagella, which impedes normal flagellar beating.

Abstract

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo–electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

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