Mechanisms and developmental functions of cytoplasmic flows in early embryogenesis

早期胚胎发生中细胞质流动的机制和发育功能

基本信息

批准号：
10796050
负责人：
Stefano Di Talia
金额：
$ 20.6万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10796050
关键词：
9 year old Actomyosin Address Affect Anterior Apical Biochemical Biological Biological Process Biophysics Blastoderm CDC2 gene Cell Cycle Cell Cycle Regulation Cell Nucleus Cell model Cells Characteristics Color Complex Computer software Computing Methodologies Confocal Microscopy Coupling Cullin 5 Protein Cytoplasm Cytoplasmic streaming Cytoskeleton Cytosol Data Detection Development Diffusion Drosophila genus Embryo Embryonic Development Endowment Ensure Epithelium Event Feedback Fertilization Fluorescence Recovery After Photobleaching Funding Gel Generations Genes Genetic Genetic Diseases Geometry Giant Cells Goals Hour Human Image Lasers Learning Link Liquid substance Location Mammals Measures Mechanics Methodology Methods Microscope Mitotic Modeling Molecular Morphogenesis Nature Normal Cell Nuclear Outcome Pattern Phase Play Positioning Attribute Process Property Protein phosphatase Proteins Role Scanning Signal Transduction System Technology Temperature Testing Time Tissues Transgenic Organisms Viscosity Work biological systems biophysical model constriction detector embryo cell experimental study improved in vivo insight mathematical model mechanical signal morphogens mutant novel optogenetics organ growth parent grant physical process physical property programs quantitative imaging spatiotemporal ubiquitin ligase

项目摘要

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.

抽象的父授予的目的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，但显微镜为9 年龄，显示出衰落的迹象。一个更可靠和改进的显微镜，它利用了优势在检测技术的最新进展中，我们的工作将需要保持竞争力。因此，我们是请求资金购买Leica Stellaris。我们的显微镜在工作日每天使用大约16个小时在周末大约6个小时/天。我们所有的项目都以实时成像为中心，需要高灵敏度，强调具有最新检测器的显微镜的重要性。我们进行多色成像并生成许多转基因线，这些系在整个可见光谱上表达探针，因此我们需要6 激光线。我们还进行光漂白（FRAP）实验后进行光遗传学和荧光恢复这证明了对激光扫描系统的需求和Leica的专业软件。一个新的显微镜对于实现父赠款的目标至关重要，这是在下面概述的。生化和机械信号的整合是生物学的重要且无处不在的特征系统。在胚胎开发过程中，复杂的组织组织和功能。我们已经表明，在果蝇胚胎发生的早期，前芽剂阶段，细胞周期振荡器和肌动球蛋白收缩力控制核定位。这种机制的核心是细胞质皮质收缩引发的流动。这些反过有丝分裂细胞周期蛋白依赖性激酶1（CDK1）和蛋白磷酸酶1（PP1）。这些流动核和负责它们在整个胚胎上的准确定位。该提议的目的是建立在这些基础上发现并更深入地了解细胞质流的机制和发育功能。我们将采用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化和机械信号的耦合以及细胞质。细胞骨架和细胞质之间的耦合将通过两流体模型建模：活性收缩凝胶和粘性细胞质。 2。我们将使用遗传和光遗传学方法来改变皮质收缩力以及转基因方法改变胚胎的几何形状和一种新颖的设置以控制温度。这些实验将提供有关生成的分子机制的见解以及细胞质流的特性。 3。我们将测试细胞质流是否在形成中起作用形态学梯度。具体而言，我们将使用定量成像和数学建模来确定细胞质流是否影响双形形态学前后梯度的形成果蝇果蝇胚胎。总之，这些研究将为整合提供一个新的范式可能与其他发育系统具有一般相关性的生化和机械信号。