Mechanisms underpinning meiotic spindle formation and behavior

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Administrative Supplement for R35 GM142537 (PD/PI: Balboula): “Mechanisms underpinning meiotic spindle formation and behavior” From Parent R35 GM142537 Project Summary: Meiosis is a specialized set of cell divisions that produce haploid gametes. During meiosis I (MI) in females, bipolar spindle formation and positioning within the oocyte must be regulated tightly to ensure faithful chromosome segregation and proper genome inheritance. In somatic mitotic cells, bipolar spindle formation and positioning rely on a centrosome pair, each of which contains two centrioles. Interestingly, meiotic oocytes lack centrioles and, hence, lack classic centrosomes. Meiotic oocytes, instead, contain numerous microtubule (MT) organizing centers (MTOCs) that are organized, by largely unknown mechanisms, to establish two spindle poles (polar MTOCs). The traditional view was that, in mammalian oocytes, MTs (and their associated proteins) are the only cytoskeletal components responsible for organizing such MTOC spindles. However, recent data suggest that F-actin is also involved in spindle bipolarity regulation. How F-actin interacts with MTs to regulate polar MTOC organization during MI represents a critical gap in our understanding of how the meiotic spindle is built. We recently identified a novel, functionally different, class of MTOCs (mcMTOCs) and found that spindle maintenance at the oocyte center is regulated by two opposing forces (mcMTOC-mediated MTs vs. F-actin). We also recently observed that ~50% of spindles are not assembled centrally. To date, such peripheral spindle assembly was unobservable owing to technical limitations associated with spindle fluorescence (i.e. live imaging). To circumvent this, we generated a Cep192-eGfp reporter mouse model enabling spindle tracking wherever it is assembled. Strikingly, peripheral spindle formation is typically followed by spindle migration towards the center – a previously undocumented phenomenon. Understanding the molecular mechanisms regulating this corrective developmental event represents a major gap in our knowledge of meiotic spindle spatiotemporal regulation during MI. This proposal lays the foundations for our long-term goal: To understand how two critical events during MI — bipolar spindle assembly and positioning — are regulated, in the absence of centrioles, to ensure faithful chromosome segregation. To do so, we will utilize state-of-the-art approaches, including transgenic mouse models, genetic constructs, laser ablation, and cutting-edge imaging, to tackle three critical goals: (i) determine how F-actin interacts with MTs to organize polar MTOCs during bipolar spindle building, (ii) establish the mechanism(s) by which the peripheral acentriolar spindle migrates to the oocyte center, and (iii) determine whether differences in biochemical compositions of mcMTOCs vs. polar MTOCs underlie their functional differences. Given that chromosome segregation errors (very common during MI) lead to aneuploidy, the leading genetic cause of developmental disorders and miscarriage, these studies have the potential to significantly advance our basic understanding of two fundamental processes — spindle formation and positioning — during MI whilst simultaneously shedding light on why MI is notoriously error prone.