Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes

Abstract

Cell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1-3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but-unlike mitosis-not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes.

A Gradient in Metaphase Tension Leads to a Scaled Cellular Response in Mitosis

“…(Miller et al, 2016). Recent data have shown that the primary role of Aurora B during error correction in meiosis I is the creation of kinetochore detachments, especially on chromosomes that show reduced inter-kinetochore distances, and that spindle assembly checkpoint activation occurs downstream of the detachments (Vallot et al, 2017). Our data suggest that a similar process operates in mitosis, and we build on this model to demonstrate that the error detection process may originate as a result of highly sensitive tension sensing by the cell.…”

“…Outwardly directed forces from Kinesin-5 motors are transmitted to the chromosomes via kinetochore microtubules, leading to stretching of the inter-kinetochore chromatin spring ( Figure 1D, top) and giving rise to an inwardly directed tension ( Figure 1B). Therefore, to modulate tension, we directed our efforts at Kinesin-5 molecular motors, the active source of the forces against which tension is generated (similar to previous work [Vallot et al, 2017]). We reasoned that by targeting outward force generation by Kinesin-5 motors, tension could be modulated without disruptions to chromosome structure or replication, alterations to kinetochore structure, or suppression of kinetochore microtubule dynamics, which would provide a powerful method for quantitatively evaluating the sensitivity of the cell to tension as a mechanical signal during mitosis.…”

Post‐metaphase correction of aberrant kinetochore‐microtubule attachments in mammalian eggs

“…We have used a protocol described in [Ref. ], where cells undergo cold treatment to remove less stable microtubules that are not attached to the kinetochores in a stabilizing buffer instead of PBS as used by [Ref. ], to preserve MTs.…”

“…Microtubules were stabilized by fixation in 1.9% formaldehyde in BRD80 buffer (80 mM K‐PIPES, 1 mM MgCl 2 , 1 mM EGTA, pH 6.8) after 5 min cold treatment in 80 mM K‐PIPES, 1 mM MgCl 2 , pH 7.4, as described in [Ref. ]. Prometaphase II oocytes were fixed 1.5–2.5 h after the first polar body extrusion.…”

Multiple Determinants and Consequences of Cohesion Fatigue in Mammalian Cells

“…At high concentrations, STLC induces collapse of spindle poles. But at reduced concentrations, spindles can be maintained with decreased interpolar distance and spindle tension (Vallot et al, 2017). The decrease in spindle tension would be expected to reduce the outward force on kinetochores.…”