Mechanisms and developmental functions of cytoplasmic flows in early embryogenesis

摘要 该项目的目标是阐明细胞质流在早期胚胎发生中的作用。对于这项工作， 共聚焦显微镜起着至关重要的作用。我们目前拥有Leica SP8显微镜，但显微镜是9 岁，并显示出衰退的迹象。一种新的更可靠和改进的显微镜，它利用了 我们的工作要保持竞争力，就需要了解探测技术的最新进展。因此，我们 申请资金购买一台徕卡Stellaris我们的显微镜在工作日每天使用约16小时 周末每天约6小时。我们所有的项目都以实时成像为中心，需要高灵敏度， 强调了拥有一台配备最新探测器的显微镜的重要性。我们做多色成像 并产生许多转基因株系，其在整个可见光谱内表达探针，因此我们需要6个 激光线我们还进行光遗传学和光漂白后荧光恢复（FRAP）实验 这证明了对徕卡激光扫描系统和专用软件的需求。一个新的显微镜 对于实现下文概述的父母补助金目标至关重要。 生物化学和机械信号的整合是生物学的一个重要和普遍存在的特征， 系统.在胚胎发育过程中，这种整合是复杂组织组织所必需的， 功能我们已经证明，在果蝇胚胎发生的早期，前胚盘阶段， 振荡器和肌动球蛋白收缩控制核定位。这种机制的核心是细胞质 由皮层收缩引发的血流。这些，反过来，在时空上与 有丝分裂细胞周期蛋白依赖性激酶1（Cdk 1）和蛋白磷酸酶1（PP 1）。这些流动输送核， 负责它们在胚胎中的准确定位。本提案的目标是在这些基础上 研究结果，并更深入地了解细胞质流动的机制和发育功能。我们 将采取三种方法来解决这些基本问题。1.我们将建立一个生物物理模型， 捕获生物化学和机械信号的耦合以及生物分子的有效物理性质。 细胞质细胞骨架和胞质溶胶之间的耦合将通过双流体模型来建模： 活性收缩凝胶和粘性胞质溶胶。2.我们将使用遗传学和光遗传学的方法来改变大脑皮层 收缩性以及转基因方法来改变胚胎的几何形状和一种新的设置来控制 温度这些实验将提供对产生的分子机制的见解 和细胞质流动的特性。3.我们将测试细胞质流动是否在细胞质的形成中发挥作用。 形态梯度具体来说，我们将使用定量成像和数学建模来确定 细胞质流动是否影响Bicoid形态发生前后梯度的形成， 合胞的果蝇胚胎。这些研究将提供一个新的范式， 生物化学和机械信号可能与其他发育系统具有普遍相关性。

ABSTRACT The goal of the parent grant is to elucidate the role of cytoplasmic flows in early embryogenesis. For this work, confocal microscopy plays a crucial role. We currently own a Leica SP8 microscope, but the microscope is 9 years old and it shows signs of decline. A new more reliable and improved microscope, which takes advantage of recent progress in detection technologies, will be needed for our work to remain competitive. Thus, we are requesting funds to purchase a Leica Stellaris. Our microscope is used about 16 hours/day during the weekdays and about 6 hours/day during weekends. All our projects are centered on live imaging and require high sensitivity, highlighting the importance of having a microscope with the most up-to-date detectors. We do multi-color imaging and generate many transgenic lines which express probes across the entire visible spectrum, thus we need 6 laser lines. We also perform optogenetic and Fluorescence Recovery After Photobleaching (FRAP) experiments which justify the need for a laser scanning system and a specialized software from Leica. A new microscope would be crucial to achieve the goals of the parent grant, which are outlined below. The integration of biochemical and mechanical signals is an important and ubiquitous feature of biological systems. During embryonic development, this integration is required for complex tissue organization and function. We have shown that in the early, pre-blastoderm stages of Drosophila embryogenesis the cell cycle oscillator and actomyosin contractility control nuclear positioning. At the core of this mechanism are cytoplasmic flows that are initiated by cortical contractions. These, in turn, are linked spatiotemporally to the oscillation of mitotic Cyclin-dependent kinase 1 (Cdk1) and protein phosphatase 1 (PP1). These flows transport nuclei and are responsible for their accurate positioning across the embryo. The goal of this proposal is to build on these findings and to understand more deeply the mechanisms and developmental functions of cytoplasmic flows. We will take three approaches to address these fundamental questions. 1. We will build a biophysical model that captures the coupling of biochemical and mechanical signals and the effective physical properties of the cytoplasm. The coupling between the cytoskeleton and the cytosol will be modeled by a two-fluid model: an active contractile gel and a viscous cytosol. 2. We will use genetic and optogenetics approaches to alter cortical contractility as well as transgenic approaches to change the geometry of the embryo and a novel setup to control temperature. These experiments will provide insight on the molecular mechanisms underlying the generation and the properties of cytoplasmic flows. 3. We will test whether cytoplasmic flows play a role in the formation of morphogen gradients. Specifically, we will use quantitative imaging and mathematical modeling to determine whether cytoplasmic flows affect the formation of the anterior-posterior gradient of Bicoid morphogen in the syncytial Drosophila embryo. Taken together these studies will provide a new paradigm for the integration of biochemical and mechanical signals that is likely to have general relevance for other developmental systems.