Project Summary/Abstract Localization of mRNAs to specific cells of an embryo, or to sub-cellular regions in the cell, enables spatially controlled protein production. Distinct concentrations of proteins lead to differential cell fate determination, body patterning, and cell function during development in many animals. Especially, this mechanism of localized translation appears essential in embryonic cells that are large and that undergo rapid cell divisions, requiring immediate input of specific protein without relying on diffusion kinetics from translation elsewhere in the cell. Indeed, the mitotic spindle is a sub-cellular region where localization of mRNAs has been reported in eggs/ embryos of various organisms. The mechanism or process of localized translation on the spindle has been, however, little identified. The proposed research focuses on a conserved DEAD-box RNA helicase Vasa that is localized on the spindle of every blastomere during embryogenesis. We previously identified Vasa functions in general translational regulation and its dysfunction results in ~80% reduction of protein synthesis and mitotic defects in the sea urchin embryo. We, therefore, hypothesize in these embryonic cells, Vasa contributes to the process of localized mRNA translation on the spindle. To test our hypothesis, using sea urchin embryos that show constant and robust Vasa expression on the spindle of every blastomere, we will visualize in vivo and in real time localized translation and validate how each mRNA/protein is differentially regulated at each sub-cellular region during embryogenesis. We will then identify sub-cellular function of Vasa by manipulating its localization in the cell using advanced imaging techniques such as optogenetics and Chromophore- Assisted Light Inactivation (CALI). A biological event of localized mRNA translation as well as a DEAD- box RNA-helicase Vasa is highly conserved among various organisms and cells. The outcomes of this project will be thus useful to all researchers, especially those who study how sub-cellular level of mRNA and protein regulation impacts cellular regulation and development in any cells and organisms.