Sample preparation

Samples for electron tomography were prepared as described (Woog et al., 2012). Briefly, hermaphrodites were dissected in Minimal Edgar’s Growth Medium (Edgar, 1995) and embryos in early meiosis were selected and transferred to cellulose capillary tubes (Leica Microsystems, Vienna, Austria) with an inner diameter of 200 μm. The embryos were observed with a stereomicroscope, transferred to membrane carriers at appropriate stages and immediately cryo-immobilized using an EMPACT2 high-pressure freezer (Leica Microsystems, Vienna, Austria) equipped with a rapid transfer system (Pelletier et al., 2006). Freeze substitution was performed over 3 d at −90°C in anhydrous acetone containing 1% OsO₄ and 0.1% uranyl acetate using an automatic freeze substitution machine (EM AFS, Leica Microsystems, Vienna, Austria). Epon/Araldite infiltrated samples were then embedded in a thin layer of resin and polymerized for 3 d at 60°C. Embedded embryos were re-mounted on dummy blocks and serial semi-thick (300 nm) sections were cut using an Ultracut UCT Microtome (Leica Microsystems, Vienna, Austria). Sections were collected on Formvar-coated copper slot grids and post-stained with 2% uranyl acetate in 70% methanol followed by Reynold’s lead citrate.

Electron tomography

For dual-axis electron tomography (Mastronarde et al 1997), 15-nm colloidal gold particles (Sigma-Aldrich) were attached to both sides of semi-thick sections to serve as fiducial markers for subsequent image alignment. Series of tilted views were recorded using a TECNAI F30 transmission electron microscope (FEI Company, Eindhoven, The Netherlands) operated at 300 kV. Images were captured every 1.0° over a ± 60° range at a pixel size of 2.3 nm using a Gatan US1000 2K x 2K CCD camera. Using the IMOD software package, a montage of 2 x 1 [meiosis I: metaphase #1, metaphase #2, anaphase (late) #1, anaphase (late) #2; meiosis II: metaphase #1, metaphase #2] or 2 x 2 [meiosis I: anaphase (early); meiosis II: anaphase (late)] frames was collected and combined for each serial section to cover the lengths of the meiotic spindles (Kremer et al., 1996; Mastronarde, 1997).

For image processing the tilted views were aligned using the positions of the fiducials. Tomograms were computed for each tilt axis using the R-weighted back-projection algorithm (Gilbert, 1972). In order to cover the entire volume of each spindle, we acquired tomograms of about 8-12 consecutive sections per sample. In total, we recorded 10 wild-type spindles in meiosis I and II (Supplementary Table 1 and 2).

Three-dimensional reconstruction and automatic segmentation of microtubules

We used the IMOD software package (http://bio3d.colorado.edu/imod) for the calculation of electron tomograms (Kremer et al., 1996). We applied the Amira software package for the segmentation and automatic tracing of microtubules (Stalling et al., 2005). For this, we used an extension to the filament editor of the Amira visualization and data analysis software (Redemann et al., 2017; Redemann et al., 2014; Weber et al., 2012). We also used the Amira software to stitch the obtained 3D models in z to create full volumes of the recorded spindles (Redemann et al., 2017; Weber et al., 2014). The automatic segmentation of the spindle microtubules was followed by a visual inspection of the traced microtubules within the tomograms. Correction of the individual microtubule tracings included: manual tracing of undetected microtubules, connection of microtubules from section to section and deletions of tracing artifacts (e.g. membranes of vesicles). Approximately 5% of microtubules needed to be corrected (Redemann et al., 2017).