C. Elegans meiotic spindles
Redemann, Stefanie (2021), C. Elegans meiotic spindles, Dryad, Dataset, https://doi.org/10.5061/dryad.x3ffbg7k5
The female meiotic spindles of most animals are acentrosomal and undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of acentrosomal spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown.
To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic C. elegans spindles undergoing the transition from metaphase to anaphase. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over on second time scales. The results show that the transition from metaphase to anaphase correlates with an increase in the number of microtubules and a decrease in their average length. Detailed analysis of the tomographic data revealed that the length of microtubules changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for those microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the observed large-scale reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those that are closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe alone. In anaphase, even microtubules close to the chromosomes show no signs of cutting. This suggests that the most prominent drivers of spindle rearrangements from metaphase to anaphase are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on the presence of katanin.
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.
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).
Data can be opened using the Amira software from FEI
Deutsche Forschungsgemeinschaft, Award: MU 1423/3-1
Technische Universität Darmstadt, Award: Frauenhabilitation
Natural Sciences and Engineering Research Council of Canada
National Science Foundation, Award: DMR-0820484
National Institutes of Health, Award: 1R01GM104976-01
Human Frontier Science Program, Award: RGP 0034/201