MAPK3/1 (ERK1/2) and Myosin Light Chain Kinase in Mammalian Eggs Affect Myosin-II Function and Regulate the Metaphase II State in a Calcium- and Zinc-Dependent Manner1

Abstract

Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

MRCK controls myosin II activation in the polarized cortex of mouse oocytes and promotes spindle rotation and male pronucleus centration

“…Intriguingly, we recently uncovered that the active myosin II ring is lost upon Cdc42 inactivation (Dehapiot et al, 2021). Therefore, while the current view is that the myosin II ring is driven by myosin light chain kinase/MLCK (Deng et al, 2005; Matson et al 2006; Deng et al, 2007; Ajduk et al, 2011; McGinnis et al, 2015; Mackenzie et al, 2016), our observations rather support a Cdc42-dependent mechanism. In line with this view, we previously reported that Cdc42-inhibited MII oocytes do not experience spindle drift, despite a loss of the actin cap (Dehapiot et al, 2013; Dehapiot and Halet, 2013).…”

“…These results are consistent with a loss of actomyosin contractility in the polarized cortex of MRCK-inhibited oocytes. Importantly, these data also demonstrate that inactivation of ring myosin II is not associated with a drift of the MII spindle away from the cortex, contrary to previous studies using ML-7 (Deng et al, 2005;Deng et al, 2007;McGinnis et al, 2015).…”

“…In this regard, apical constriction of endoderm precursor cells in C. elegans gastrulation was initially reported to rely on MLCK for myosin II activation, based on the use of ML-7, but was later found to actually rely on MRCK (Lee and Goldstein, 2003; Marston et al, 2016). In mouse oocytes, conflicting results have been reported as to whether ML-7 inhibits PB2 emission (Matson et al, 2006; Larson et al, 2010; Ajduk et al, 2011; Wang et al, 2011; McGinnis et al, 2015). Moreover, oocyte-specific invalidation of the Mylk1 gene, which encodes smooth muscle and nonmuscle MLCK, suggested that MLCK is dispensable for oocyte maturation, fertilization and polar body emission (Liang et al 2015).…”

Keratin 6 regulates collective keratinocyte migration by altering cell–cell and cell–matrix adhesion

“…Because it took 1 h to set up imaging, the videos and quantifications represented the effect of 1-h pretreatment with each drug and 16-h migration in the presence of each drug. Phase-contrast imaging (McGinnis et al, 2015) was performed using an AxioObserver Z1 microscope equipped with an incubation chamber (37°C and 5% CO 2 ), a Zeiss EC Plan Neofluar 10×/0.3 Ph1 objective, and AxioVision software at 10-min intervals for 16 h. The migration area was calculated by subtracting the area measured at time 0 from the area measured after 16 h. PIV analysis of live-cell phase-contrast time-lapse videos was performed using MatLab as described (Weiger et al, 2013; Lee et al, 2016). Two interrogation window sizes (64 × 64 and 32 × 32 pixels) were used to segment the images.…”

Micropipette Aspiration of Oocytes to Assess Cortical Tension

“…If desired, oocytes can be loaded with DAPI to label the maternal DNA. Our method is based on what was originally reported for studies of sperm–egg fusion [32], incubating oocytes in culture medium containing 1 μg/mL DAPI for 90 min, followed by washing the oocytes through three drops of culture medium [33]. Alternatively oocytes can be microinjected with mRNA encoding a fluorescently tagged histone (e.g., H2B-mCherry [34, 35]).…”