Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Abstract

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/ C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast

“…Second, the meiosis II-specific process is triggered by the appearance of an essential activator, such as Cdk1-Clb, which is linked to the onset of M-phase, but is not necessarily specific to meiosis II. An additional requirement for the activity of APC/C Cdc20 would allow the control of events specific to anaphase II, such as PSM formation (Fig 9B) or the deprotection of centromeric cohesin (Mengoli et al, 2021).…”

“…In the main text, all green and red fluorescent proteins are abbreviated as GFP and RFP, respectively. We used CDC20-mAR strains for arrest/ release at metaphase I (Arguello-Miranda et al, 2017) and cdc20 ts -mAR strains for arrest at metaphase II (Mengoli et al, 2021). Estradiol-inducible expression from the GAL promoter (called P EST herein) was achieved with a Gal4-estrogen receptor fusion controlled by the GPD1 promoter (Benjamin et al, 2003).…”

“…Imaging was performed as described (Mengoli et al, 2021). Meiotic cells were applied to an 8-well glass-bottom µ-slide (ibidi, Gr€ afelfing, Germany) coated with Concanavalin A in 250 µl of SPM per well to give a density of 20-30 cells per field of view.…”

“…By contrast, it is unclear how meiosis II-specific events are regulated. These include, for instance, the reduplication of spindle pole bodies (SPBs, the yeast centrosomes), the initiation of spore differentiation, translation of the Clb3 cyclin, redistribution of mitochondria, and deprotection of centromeric cohesin (Carlile & Amon, 2008;Neiman, 2011;Berchowitz et al, 2013;Agarwal et al, 2018;Sawyer et al, 2019;Mengoli et al, 2021). While these events depend on the Hrr25 or the Ime2 kinase, little is known about the regulation of these kinases (Benjamin et al, 2003;Petronczki et al, 2006).…”

Precise and Dynamic Temperature Control in High-Resolution Microscopy with VAHEAT

“…It is particularly suitable for inducing heat shock and simultaneous imaging of the sample. This approach was used for studying meiosis with temperature-sensitive mutants in yeast [5]. Other examples include calcium imaging in heat-sensitive neurons (unpublished), time-lapse imaging of thermophilic microorganisms [6], and the study of cell biology of Archaea, which is a group of organisms containing many extremophiles that are notoriously hard to image live [7].…”