Coordination of the cell cycle and the cytoskeleton during oocyte meiosis
by Ivan Avilov
Date of Examination:2023-06-01
Date of issue:2023-06-29
Advisor:Dr. Peter Lenart
Referee:Prof. Dr. Henning Urlaub
Referee:Dr. Melina Schuh
Referee:Dr. Ufuk Günesdogan
Persistent Address:
http://dx.doi.org/10.53846/goediss-9941
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Abstract
English
Meiosis serves to reduce ploidy, to half the genetic content of germ cells before they unite again at fertilization. This is achieved by two subsequent divisions called meiosis I and II without an intermittent S-phase. In oocytes, the meiotic divisions are extremely asymmetric and characterized by the emission of small polar bodies. Mos is a conserved kinase that is exclusively expressed in the oocytes of most animal species and is degraded after fertilization. Mos activates the ERK/MAPK pathway through the phosphorylation of MEK, that in turn phosphorylates MAPK. During meiosis, activation of Mos-MAPK mediates important functions: it is rendering the meiotic divisions highly asymmetric and it regulates the cell cycle machinery to suppress S-phase in between the two meiotic divisions. Thus, Mos can be seen as a universal switch between meiotic and mitotic types of cell division. However, the molecular mechanisms that underlie these highly conserved functions mediated by Mos-MAPK remain poorly understood. In my work, I used the well-established and specific inhibitor, U0126 to block the Mos-MAPK pathway in starfish oocytes. I showed that U0126-treated starfish oocytes display mitotic-like spindle phenotype during metaphase I, followed by an exit from the meiotic program instead of undergoing meiosis II. This indicates that Mos-MAPK activity at metaphase I triggers key meiotic events: the second division without an S-phase and it also ensures the asymmetry of division. To identify the downstream molecular and cellular mechanisms, I then combined phospho-proteomic profiling of Mos-MAPK-inhibited oocytes with high-resolution live-cell imaging. Thereby, I was able to correlate changes in spindle morphology and cell cycle progression with the phosphorylation of specific proteins. This led me to discover that, firstly, Mos-MAPK is responsible for the stimulation of Cyclin B synthesis through inhibition of the CPEB module, known to suppress Cyclin B translation. I have shown that injection of active Cdk1-Cyclin B complex at the meiosis I-to-II transition rescues Mos-MAPK inhibition and restores meiosis II. This indicates that Mos-MAPK-dependent synthesis of Cyclin B at the meiosis I- to-II transition is indeed sufficient to drive meiosis II. Rapid reactivation of Cdk1-Cyclin B prevents the interphase entry and therefore allows skipping the S-phase, leading to the reduction of genomic content. Secondly, phospho-proteomic profiling identified multiple proteins under the control of Mos-MAPK that are involved in the regulation of the microtubule and actin cytoskeleton. These included proteins responsible for the recruitment and organization of the pericentriolar material at centrosomes as well as proteins that nucleate and regulate the growth of astral microtubules. Mos-MAPK-dependent regulation of these proteins explains how Mos-MAPK suppresses astral microtubules, which in turn enables the positioning of the meiotic spindle to the cortex and thereby the asymmetric meiotic divisions. Besides the regulation of meiosis by Mos-MAPK, I studied the mechanisms of nuclear envelope breakdown (NEBD) in the unusually large nuclei of oocytes. In starfish, nuclear membrane fragmentation is dependent on the Arp2/3-nucleated F-actin ‘shell’. By using super-resolution microscopy, I contributed to revealing the mechanisms by which this F-actin shell ruptures the nuclear membrane. Firstly, I found that the nuclear membrane separates into pore-free and pore-dense regions. Then, F- actin nucleates within the lamina under the pore-free membrane and forms filopodia-like spikes toward the cytoplasm. These spikes protrude and rupture the pore-free nuclear membrane, converting the pore- dense membrane regions into an ER-like network that is associated with the still-intact lamina. Taken together, in my PhD project I identified the molecular mechanisms of how the second meiosis and the asymmetric divisions are controlled by Mos-MAPK in oocytes. I expect that the identification of the conserved molecular modules controlling meiotic functions in starfish will have important general implications for understanding meiosis in other species including humans.
Keywords: oocytes, meiosis, cytoskeleton. cell cycle, Mos, MAPK