Mechanisms and developmental functions of cytoplasmic flows in early embryogenesis

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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.