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Yolk granule fusion and microtubule aster formation regulate cortical granule translocation and exocytosis in zebrafish oocytes [1]

['Shayan Shamipour', 'Institute Of Science', 'Technology Austria', 'Klosterneuburg', 'Department Of Molecular Life Sciences', 'University Of Zurich', 'Zurich', 'Laura Hofmann', 'Irene Steccari', 'Roland Kardos']

Date: 2023-06

Dynamic reorganization of the cytoplasm is key to many core cellular processes, such as cell division, cell migration, and cell polarization. Cytoskeletal rearrangements are thought to constitute the main drivers of cytoplasmic flows and reorganization. In contrast, remarkably little is known about how dynamic changes in size and shape of cell organelles affect cytoplasmic organization. Here, we show that within the maturing zebrafish oocyte, the surface localization of exocytosis-competent cortical granules (Cgs) upon germinal vesicle breakdown (GVBD) is achieved by the combined activities of yolk granule (Yg) fusion and microtubule aster formation and translocation. We find that Cgs are moved towards the oocyte surface through radially outward cytoplasmic flows induced by Ygs fusing and compacting towards the oocyte center in response to GVBD. We further show that vesicles decorated with the small Rab GTPase Rab11, a master regulator of vesicular trafficking and exocytosis, accumulate together with Cgs at the oocyte surface. This accumulation is achieved by Rab11-positive vesicles being transported by acentrosomal microtubule asters, the formation of which is induced by the release of CyclinB/Cdk1 upon GVBD, and which display a net movement towards the oocyte surface by preferentially binding to the oocyte actin cortex. We finally demonstrate that the decoration of Cgs by Rab11 at the oocyte surface is needed for Cg exocytosis and subsequent chorion elevation, a process central in egg activation. Collectively, these findings unravel a yet unrecognized role of organelle fusion, functioning together with cytoskeletal rearrangements, in orchestrating cytoplasmic organization during oocyte maturation.

Here, we show that the accumulation of Cgs at the oocyte surface and their acquisition of exocytosis competency upon GV breakdown (GVBD) are driven by the concerted activities of Yg fusion and compaction and microtubule network rearrangements, respectively. Yg fusion and compaction towards the oocyte center function in this process by inducing radially outward cytoplasmic flows that lead to the translocation and accumulation of Cgs at the oocyte surface. The microtubule network, in contrast, triggers accumulation of Rab11-positive vesicles at the oocyte surface by reorganizing into acentrosomal aster-like structures that collectively translocate towards the oocyte surface and take along Rab11-positive vesicles. Finally, the decoration of Cgs by Rab11 at the oocyte surface confers competency to Cgs to be exocytosed during mature oocyte/egg activation.

To tackle this question, we turned to the last stage of zebrafish oogenesis (oocyte maturation), during which cytoplasmic reorganizations are accompanied by changes in organelle shape, size, and position, preparing the oocyte for fertilization and embryonic development [ 26 ]. Zebrafish oogenesis constitutes a 5-stage process: During stages I and II, the oocyte animal-vegetal (AV) axis becomes determined through the vegetal pole localization of the Balbiani body, a membrane-less structure rich in mitochondria, proteins, and mRNAs required for the vegetal pole establishment [ 7 , 27 ]. Stages II and III mark the formation of both cortical granules (Cgs) and yolk granules (Ygs). Cgs are exocytosed upon fertilization to induce chorion elevation, thus preventing the entry of additional sperm in to the oocyte and protecting it against physical damage [ 28 ], while Ygs function as energy reservoirs for subsequent embryonic development [ 26 ]. Stage III oocytes remain arrested in prophase I until oocyte maturation begins. During oocyte maturation (stage IV), a multitude of transitions take place within the oocyte. For instance, the GV migrates to and breaks down at the animal pole of the oocyte, which is then followed by ooplasm, the oocyte cytoplasm, accumulating at the animal pole, forming a yolk-free blastodisc. Concomitantly, Cgs obtain exocytosis competency, thus preparing the mature oocyte/egg for fertilization [ 26 , 29 – 32 ]. How these different aspects of cytoplasmic reorganization are orchestrated and spatiotemporally controlled is still largely unknown.

Cytoplasmic organization can occur in the absence of external cues, suggesting that the cytoplasm is capable of self-organization [ 13 , 14 ]. Previous research has highlighted an important role for the cell cytoskeleton, and especially the microtubule and actin networks, in driving such cytoplasmic self-organization [ 15 – 17 ]. For instance, microtubules and the movement of motors along microtubule tracks can power cytoplasmic flows and the repositioning of microtubule asters by generating viscous drag forces to the surrounding cytoplasm [ 15 , 18 – 21 ]. Likewise, myosin II–dependent contractions of both cortical and bulk actin networks can result in large-scale actomyosin network flows, which, in turn, drag the adjacent cytoplasm via friction forces acting at their interface [ 16 , 22 , 23 ]. In addition to these motor-dependent processes, actin polymerization on the surface of organelles can drive organelle motility, thereby generating active diffusion within the bulk of the cytoplasm [ 23 – 25 ]. However, to what extent cellular processes other than cytoskeletal rearrangements, such as dynamic fusion and splitting of organelles, also function in cytoplasmic reorganization, remains unclear.

Oogenesis marks the very first step in development, establishing the maternal blueprint for embryonic patterning. During this process, the oocyte grows in size by acquiring maternally provided material and completes its first meiosis to eventually become arrested in the metaphase of meiosis II until fertilization occurs [ 1 ]. Central to oogenesis is the accurate positioning of large organelles, such as the oocyte nucleus (germinal vesicle (GV)) and meiotic spindle, but also small fate-determining molecules, such as mRNAs and proteins, within the oocyte, a process fundamental for embryonic axis formation and cell fate specification [ 2 – 12 ]. Yet, how such positioning of cytoplasmic components is orchestrated in space and time is still only poorly understood.

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002146

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