Efflux Transporters in Sea Urchin Development

海胆发育中的外排转运蛋白胚胎中的外排转运蛋白具有管家和发育作用。在胚胎发生过程中，管家作用可以保护胚胎免受外部毒物的侵害。发育作用可以将内源性信号分子泵出细胞以影响相邻细胞或以自分泌方式反馈细胞本身。初步研究表明，ABC外排转运蛋白mrp家族的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受精后含有这些转运蛋白的囊泡向卵表面移动并将蛋白质插入膜中。拟议的实验将利用抗体跟踪运输蛋白位置的任何变化来证明或反驳囊泡运动假说。初步结果显示了几种发育作用。一个是在卵裂期参与细胞分裂。拟议的研究将通过注射抗体到转运蛋白的活性部位来检查效果的特异性。如果指定为特定效果，则搜索由转运体外排的活性物质。一个可能的内源性底物是白三烯，这些化合物的水平将在卵裂和后来的发育过程中测量。另一种转运蛋白功能是在初代间充质细胞分化过程中，可能在外胚层和下层间充质细胞之间的相互作用中发挥信号传导作用。拟议的研究将描述新转运蛋白在发育过程中的表达，通过原位杂交对其进行定位，并利用morpholinos评估它们在特异性阻止新转运蛋白表达方面的作用。这项工作也拓宽了外排转运体的观点，在过去，外排转运体主要集中在癌症治疗期间逃避药物治疗方面的医学相关性上。拟议中的研究以新的方式看待这些无处不在的分子；在导致质膜重组的受精反应方面，在有丝分裂等正常细胞功能中的作用方面，在整合形态发生反应的信号作用方面。拟议中的研究对教育也有广泛的影响，因为这些实验结果中的许多可以整合到斯坦福大学入门生物学实验室的实验室练习中，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的（去年有400万次点击）海胆胚胎学网站（http://www.stanford.edu/group/Urchin/）上传播。环境研究与细胞和发育生物学的结合也吸引了大量的本科生来到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

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