Cytoplasmic reorganization in zebrafish oocytes

Overarchingly, the proposal aims at elucidating the molecular, cellular and biophysical mechanisms underlying the reorganization of cell cytoplasm using zebrafish eggs (oocytes) as an experimentally accessible assay-system to study this question. While the different components of the cell cytoplasm, such as the nucleus, organelles and cytoskeleton, have been extensively characterized over the past years, comparably little is known about how these components dynamically rearrange in space and time. Such cytoplasmic rearrangements are important for many core cellular processes, such as cell polarization, migration and division. In this project, we will study how the breakdown of the germinal vesicle, the nucleus of the immature oocyte, affects the actin and microtubule cytoskeleton in stage IV zebrafish oocytes, and how these cytoskeletal changes result in (i) the segregation of ooplasm from yolk granules along the animal- vegetal oocyte axis, and (ii) the localization of cortical granules to the oocyte circumference, two processes required for proper oocyte fertilization and subsequent embryonic development. Our preliminary data suggest that microtubule aster formation, in conjunction with yolk granule fusion, drive cortical granule relocalization towards the oocyte circumference, while actomyosin network contraction and flow cause the initial segregation of ooplasm and yolk granules along the animal-vegetal oocyte axis. For addressing the objectives and hypotheses, we will use a combination of sophisticated biophysical/imaging tools and theoretical modeling in order to obtain a mechanistic and quantitative understanding of the processes underlying ooplasmic reorganization. We expect that the findings obtained from this study will provide mechanistic insight into the processes underlying cytoplasmic rearrangements during oocyte maturation in zebrafish, and more generally, into the core principles by which the cell cytoplasm undergoes dynamic rearrangements. 1

Cells constantly reorganize their inner contents, and this is essential for processes like cell division and movement. While the cell's skeleton is known to drive much of this reorganization, much less is understood about how changing the size and position of organelles affects it. In this study, we examined how zebrafish egg cells (oocytes) prepare for fertilization. We found that special vesicles called cortical granules move to the surface of the oocyte during a key maturation step. This happens through two coordinated processes: large yolk granules fuse and pull inward, creating outward cytoplasmic flows, and microtubule structures called asters form and move toward the cell surface. We also discovered that vesicles carrying Rab11-a protein important for transport and secretion-gather with the cortical granules at the surface. Rab11-positive vesicles are transported by these microtubule asters, which form when the cell cycle regulator CyclinB/Cdk1 is released and then move outward by attaching to the actin-rich cell cortex. Finally, we show that Rab11 must be present on cortical granules for them to release their contents, a step required to lift the protective chorion around the egg. Together, these findings reveal that organelle fusion works hand-in-hand with the cytoskeleton to organize the egg cell during maturation.