Trigger waves and coupled oscillations in the embryonic cell cycle

描述(申请人提供)：最近，我们证明了CDK1/CDC25/Wee1系统的双稳性允许CDK1的激活通过所谓的触发波，即激活和失活的波，沿着轴突向下传播，使CDK1的激活通过非洲爪哇细胞质快速传播。这项工作是通过开发一种与荧光显微镜兼容的特氟龙管系统来实现的，该系统允许循环提取物进行十几个或更多完整的细胞周期而不会死亡。CDK1触发波的速度(~60�m/min)足以解释完整胚胎分裂前的有丝分裂和表面收缩波(SCW)的动力学。这项研究发表在去年的《自然》杂志上。我们建议通过分成三个具体目标的研究来加强这项工作：目标1。卵子、卵母细胞和提取物中的有丝分裂和减数分裂触发波。我们现在计划更详细地研究有丝分裂波，并研究核和中心体在这些波的产生和传播中的作用。我们还计划研究有丝分裂波之间的相互作用，我们怀疑这有助于防止CDK1激活的异位焦点破坏第一个细胞周期。最后，我们计划描述在卵母细胞中新发现的减数分裂触发波现象的机制和意义，我们怀疑它可能参与第一极体的排出和减数分裂的完成1。目的2.细胞间耦合和多细胞胚胎的同步化。一旦受精卵开始分裂，保持单个卵裂球内有丝分裂在空间上协调的问题就变得不那么成问题了，但保持不同细胞之间有丝分裂协调的问题就更成问题了。在初步研究中，我们已经表明，当胚胎与短暂的温度梯度去同步时，细胞随后恢复同步。几种机制，单独的或共同的，可以解释这种同步，包括通过细胞质桥、缝隙连接和细胞骨架元件的通信。我们计划通过在完整的胚胎和鸡蛋提取物中进行实验来测试这些想法。目的3.细胞凋亡的空间协调性。Caspase系统包括多个正反馈环路，可以产生双稳态，并有助于确保细胞凋亡是全有或全无的，并且具有不可逆性。我们计划使用非洲爪哇卵提取液作为模型系统，测试caspase激活是否实际上是双稳的。如果是这样的话，这种双稳性就有可能通过触发波使细胞的凋亡状态迅速地通过脑电传播。初步研究表明，这种情况是这样的：凋亡状态明显地以~15�m/min的恒定速度通过非洲爪哇细胞质传播。我们计划在特氟龙管提取系统中描述这些波的特征，并剖析产生它们的反馈回路。

DESCRIPTION (provided by applicant): Recently we demonstrated that the bistability of the Cdk1/Cdc25/Wee1 system allows Cdk1 activation to propagate rapidly through Xenopus cytoplasm by means of what are termed trigger waves, waves of activation and inactivation that spread the way action potentials spread down an axon. This work was made possible through the development of a Teflon tube system that is compatible with fluorescence microscopy and allows cycling extracts to carry out a dozen or more complete cell cycles without dying. The speed of the Cdk1 trigger wave (~60 �m/min) is sufficient to account for the dynamics of mitosis and of the surface contraction waves (SCWs) that occur prior to cleavage in intact embryos. This work was published last year in Nature. We propose to build upon this work through studies divided into three Specific Aims: Aim 1. Mitotic and meiotic trigger waves in eggs, oocytes, and extracts. We now plan to examine mitotic waves in greater spatial detail and examine the roles of nuclei and centrosomes in the generation and propagation of these waves. We also plan to examine the interplay between mitotic waves, which we suspect helps keep ectopic foci of Cdk1 activation from disorganizing the first cell cycle. Finally, we plan to characterize the mechanism and significance of a newly discovered meiotic trigger wave phenomenon in oocytes, which we suspect may be involved in the expulsion of the first polar body and the completion of meiosis 1. Aim 2. Intercellular coupling and the synchronization of multicellular embryos. Once the fertilized egg begins to divide, the issue of keeping mitosis spatially coordinated within individual blastomeres becomes less problematic, but the issue of keeping mitosis coordinated between separate cells becomes more problematic. In preliminary studies we have shown that when an embryo is desynchronized with a transitory temperature gradient, the cells subsequently return toward synchrony. Several mechanisms, singly or together, may explain this synchronization, including communication through cytoplasmic bridges, gap junctions, and cytoskeletal elements. We plan to test these ideas through experiments in intact embryos and egg extracts. Aim 3. Spatial coordination of apoptosis. The caspase system includes multiple positive feedback loops that could generate bistability and help ensure that apoptosis is all-or-none and irreversible in character. We plan to test whether caspase activation is, in fact, bistable, using Xenopus egg extracts as a model system. If it is, then it is possible that this bistability allows the apoptotic state to propagate rapidly through te egg via trigger waves. Preliminary studies indicate that this is the case: the apoptotic state apparently propagates through Xenopus cytoplasm at a constant speed of ~15 �m/min. We plan to characterize these waves in the Teflon tube extract system and to dissect the feedback loops that generate them.