Efflux Transporters in Sea Urchin Development

IBN-0446384外流转运蛋白在海胆发育中的项目摘要胚胎中的外流转运蛋白可以同时起到管家和发育的作用。管家的角色可以保护胚胎在胚胎发育过程中免受外部毒物的影响。发育作用可以将内源性信号分子泵出细胞，以影响邻近细胞或以自分泌方式对细胞本身进行反馈。初步工作表明，ABC外排转运体的MRP家族成员在未受精的海胆卵中处于休眠状态，并作为受精反应的一部分变得活跃。活性的增加似乎是由于含有这些转运蛋白的囊泡在受精后移动到卵子表面，并将蛋白质插入到膜中。拟议中的实验将证明或反驳囊泡运动假说，使用抗体跟踪运输蛋白位置的任何变化。初步结果表明几个发育作用。一种是在卵裂期推进细胞分裂的作用。这项拟议的研究将通过向转运蛋白的活性部位注射抗体来检验这种效果的特异性。如果指示为特定效果，则将搜索由转运体排出的活性物质。一个看似合理的内源性底物是白三烯，这些化合物的水平将在卵裂和以后的发育过程中进行测量。另一个转运蛋白功能是在初级间充质细胞分化的信号传递中发挥作用，可能是在外胚层和底层间充质细胞之间的相互作用中。拟议的研究将描述新的转运蛋白在发育过程中的表达，通过原位杂交对它们进行定位，并使用吗啡来评估它们在特定阻止新的转运蛋白表达方面的作用。这项工作也拓宽了外排转运蛋白的视野，过去主要集中在它们在抗癌治疗过程中逃避药物治疗方面的医学意义。这项拟议的研究以新的方式看待这些普遍存在的分子；从导致质膜重组的受精反应方面，从正常细胞功能(如有丝分裂)中的作用，以及整合形态发生反应的信号作用方面。这项拟议的研究也对教育产生了广泛的影响，因为许多这些实验结果可以整合到斯坦福大学生物入门实验室以及全国各地的高中和大学练习中。为了促进这一点，这项研究建议的练习将在广泛使用(去年获得400万点击量)的海胆胚胎学网站(http://www.stanford.edu/group/Urchin/)上传播。这种环境工作与细胞和发育生物学的结合也吸引了许多本科生来到P.I的实验室(通常来自代表性不足的群体)；这些学生经常因出色的本科生研究而获得大学的认可，其中许多学生后来继续攻读发育生物学和环境科学的研究生课程

Project Summary for IBN-0446384Efflux Transporters in Sea Urchin DevelopmentEfflux transporters in embryos can have both housekeeping and developmental roles. The housekeeping role can protect the embryo from external toxicants during embryogenesis. The developmental role can pump endogenous signaling molecules out of cells to influence adjacent cells or to feedback on the cell itself in an autocrine fashion. Preliminary work reveals members of the mrp family of ABC efflux transporters are dormant in the unfertilized sea urchin egg and become active as part of the response to fertilization. The increased activity appears to result from movement of vesicles containing these transporters to the egg surface after fertilization and insertion of the proteins into the membrane. Proposed experiments will prove or disprove the vesicle movement hypothesis using antibodies to follow any changes in the location of the transport proteins.Preliminary results suggest several developmental roles. One is a role in forwarding cell division during the cleavage period. The proposed research will examine specificity of the effect by injection of antibodies to the active site of the transporter. If indicated as a specific effect a search will be made for the active substance effluxed by the transporter. A plausible endogenous substrate are leukotrienes and levels of these compounds will be measured during cleavage and later development. The other transporter function is a role in signaling in primary mesenchyme cell differentiation, perhaps in the cross-talk between ectoderm and underlying mesenchyme cells. The proposed studies will describe the expression of new transporters during development, localize them by in situ hybridization and assess their role using morpholinos to specifically prevent new transport expression. This work also broadens the view of efflux transporters, which has in the past been primarily centered on their medical relevance in terms of evading drug therapy during treatment against cancer. The proposed research looks at these ubiquitous molecules in new ways; in terms of a fertilization response leading to re-organization of the plasma membrane, in terms of a role in normal cell functions such as mitosis and in terms of a signaling role for integrating morphogenetic responses. The proposed research also has broad impact on education, as many of these experimental results can be integrated into laboratory exercises used in introductory biology labs at Stanford and in high school and college exercises across the country. To facilitate this, exercises suggested by this research will be disseminated on the widely used (last year received 4 million hits) Sea Urchin Embryology web site (http://www.stanford.edu/group/Urchin/ ).This combination of environmental work with cell and developmental biology also attracts numerous undergraduate students to the P.I's lab (often from under-represented groups); these students often receive University recognition for outstanding undergraduate research and many of these students later go on to graduate study in developmental biology and environmental sciences