Supplementary Materials

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

Sudarshan Gadadhar, Gonzalo Alvarez Viar, Jan Niklas Hansen, An Gong, Aleksandr Kostarev, Côme Ialy-Radio, Sophie Leboucher, Marjorie Whitfield, Ahmed Ziyyat, Aminata Touré, Luis Alvarez, Gaia Pigino, Carsten Janke

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S10
  • Table S2
  • Captions for Tables S1 and S3
  • Captions for Movies S1 to S6
  • References
MDAR Reproducibility Checklist
Table S1
Quantification of sperm quality, swim pattern and in vitro fertilization index: Tables showing the data from individual experiments for the different motility parameters determined by CASA (Fig. 2C, fig. S4C), the sperm count (fig. S3F), sperm viability (fig. S3G), swimming behaviour (Fig. 4F), and the in vitro fertilization index (Fig. 2B, fig. S4B).
Table S3
Quantifications from analysis using SpermQ software: Tables corresponding to the SpermQ analysis of mean curvature (Fig. 3B, fig. S5A and S5B), flagellar amplitude (Fig. 3C, fig. S5D) and peak frequency (Fig. 3D, fig. S5E) of individual sperm cells from different wild-type and Ttll3-/-Ttll8-/- mice.

Images, Video, and Other Media

Movie S1
Flagellar beat of sperm tethered at their heads
Dark-field microscopy of head-tethered sperm recorded at 250 frames-per-second (fps). (A) Symmetric and asymmetric flagellar beat for wild-type and Ttll3-/-Ttll8-/- sperm, respectively (top). The symmetry of the beat is clearly shown in the corresponding color-coded flagellar beat projections (bottom panels from Fig. 3A). (B to D) Flagellar beat analyses of tethered sperm (panels from Fig. 3, B to D): Ttll3-/-Ttll8-/- sperm have a more than three times higher curvature (B) and less than half the beat amplitude (C). The beat frequency (D) is asymmetric: it is lower near the sperm head, but more than double in the rest of the flagellum.
Movie S2
Swimming behaviors of sperm
Dark-field microscopy of free-swimming sperm from wide-field images (A and B) were recorded at 50 fps. Single-sperm transitions (C to E) were recorded at 250 fps. Wild-type sperm swim almost exclusively along an elongated curvilinear path (A and C). In contrast, most Ttll3-/-Ttll8-/- sperm (~86%) swim in circular paths (B and E), with a small fraction (~14%) showing incidental progressive motility (B and D).
Movie S3
Helical and circular swimming of Ttll3-/-Ttll8-/- sperm
Dark-field microscopy of free-swimming sperm recorded at 250 fps in custom-made observation chambers of 100 μm depth. Upon reaching the wall of the observation chamber, the Ttll3-/-Ttll8-/- sperm transitions from helical to circular swimming (still panel from Fig. 4D).
Movie S4
Absence of glycylation results in altered conformations of axonemal ODA heavy chains
(A) Fitting the crystal structures of the dynein motor domain onto isosurface renderings of the 96-nm repeats in sperm axoneme shows an altered conformation of both β-heavy chain (magenta) and γ- heavy chain (green) when the dynein shifts from pre-powerstroke to the post-powerstroke condition. (B) Predominant conformations of the ODAs observed in the wild-type and Ttll3-/-Ttll8-/- sperm (panels from Fig. 6A). (C) Comparison of the transition of the ODAs shows a coordinated transition from pre-powerstroke (blue) to post-powerstroke (yellow) in the wild-type axonemes, whereas in the Ttll3-/-Ttll8-/- axonemes intermediate conformations, pre-post (orange) and post-pre (brown) are more commonly found (bottom panels from Fig. 6B).
Movie S5
IDA-f conformational reconfiguration is linked to the tilting motion of the nexindynein
regulatory complex (N-DRC) (A) Slices through class averages generated from IDA classification showing their overall transition between pre-powerstroke and post-powerstroke conformations (left). Particularly consistent rearrangement of IDA-f subspecies (encircled in red) between the two classes are shown in the right panels (from fig. S8B). (B) The conformational reconfiguration of IDA-f occurred synchronously with an opposing tilting motion of the nexin-dynein regulatory complex and the radial-spoke heads. This tilting motion was perpendicular to the MT axis and associated with a displacement of the next MT doublet. (A tub: A-tubule; B tub: B-tubule; N-DRC: nexin-dynein regulatory complex; RS: radial spoke; ODA: outer dynein arm; IDA: inner dynein arm)
Movie S6
Absence of glycylation has no apparent effect on the beating of ependymal cilia
(A) Representative video recordings of ependymal cilia acquired from 100-200-μm sagittal sections of wild-type and Ttll3-/-Ttll8-/- brain ventricles. Recordings were performed at 100 fps for 3 s using a 60× objective. The videos were slowed down 20× to scrutinize the ciliary beat waveform (one representative video in original speed is shown on the left). Visual inspection does not reveal any changes in the beat patterns of multicilia in the absence of glycylation. (B) Scatter plots showing the analysis of cilia beat frequencies from three independent experiments with each point representing an individual cell recorded from the same brain slice (number of cells are given). Median (±SEM) is indicated, p-values are determined by Student's t-test. All three experiments coherently show no differences between wild-type and Ttll3-/-Ttll8-/- cells. Note that overall values vary between different experimental days, most likely due to slight variations in imaging conditions (e.g. temperature).