A Switch in Microtubule Orientation during C. elegans Meiosis

Abstract

In oocytes of many organisms, meiotic spindles form in the absence of centrosomes [1-5]. Such female meiotic spindles have a pointed appearance in metaphase with microtubules focused at acentrosomal spindle poles. At anaphase, the microtubules of acentrosomal spindles then transition to an inter-chromosomal array, while the spindle poles disappear. This transition is currently not understood. Previous studies have focused on this inter-chromosomal microtubule array and proposed a pushing model to drive chromosome segregation [6, 7]. This model includes an end-on orientation of microtubules with chromosomes. Alternatively, chromosomes were thought to associate along bundles of microtubules [8, 9]. Starting with metaphase, this second model proposed a pure lateral chromosome-to-microtubule association up to the final meiotic stages of anaphase. Here, we applied large-scale electron tomography [10] of staged C. elegans oocytes in meiosis to analyze the orientation of microtubules in respect to chromosomes. We show that microtubules at metaphase I are primarily oriented laterally to the chromosomes and that microtubules switch to an end-on orientation during progression through anaphase. We further show that this switch in microtubule orientation involves a kinesin-13 microtubule depolymerase, KLP-7, which removes laterally associated microtubules around chromosomes. From this, we conclude that both lateral and end-on modes of microtubule-to-chromosome orientations are successively used in C. elegans oocytes to segregate meiotic chromosomes.

Local regulation of lipid synthesis controls ER sheet insertion into nuclear envelope holes to complete nuclear closure

“…1 A) and transitions into a central spindle of microtubules between the segregated chromosomes in anaphase II ("maximal spindle shortening", Fig. 1 A) (Yang et al, 2003;Fabritius et al, 2011;Redemann et al, 2018). As one set of segregated chromosomes extrudes to generate a second polar body, the haploid, oocyte-derived pronucleus forms.…”

“…As one set of segregated chromosomes extrudes to generate a second polar body, the haploid, oocyte-derived pronucleus forms. The spindle of microtubules elongate and dissipate as the oocyte-derived pronucleus forms (Yang et al, 2003;Fabritius et al, 2011;Redemann et al, 2018), but the mechanisms of remodeling and sealing the NE were not known.…”

“…Hermaphrodites were dissected in Minimal Edgar's Growth Medium (Edgar, 1995;Woog et al, 2012) and embryos in meiosis were transferred to cellulose capillary tubes (Leica Microsystems, Vienna, Austria). The embryos were observed with a stereo microscope and placed into membrane carriers at anaphase I for immediate cryoimmobilization using an EMPACT2+RTS high-pressure freezer (Leica Microsystems, Vienna, Austria) (Redemann et al, 2018). 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).…”

“…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 (Kremer et al, 1996) (http://bio3d.colorado.edu/imod), montages of 2 x 1 were collected and combined for each serial section (Redemann et al, 2018). Tomograms were computed for each tilt axis using the R-weighted back-projection algorithm (Gilbert, 1972).…”

Sumoylation regulates central spindle protein dynamics during chromosome segregation in oocytes

“…During C. elegans female meiosis, kinetochores disassemble in early anaphase I and appear to be dispensable for chromosome segregation (Dumont et al, 2010;Hattersley et al, 2016;McNally et al, 2016). In addition, tomographic reconstruction in electron microscopy of the C. elegans female meiotic spindle revealed that during anaphase I, central-spindle MTs transition from a lateral to an end-on orientation Redemann et al, 2018;Yu et al, 2019). Therefore, while the balance between central-spindle MTand kMT-driven forces may vary in different spindles, the former seems most important during female meiosis in C. elegans.…”