VPW: The Cloning Membrane Receptor-Proteins in Mammalian & Sea Urchin Development (Cell Biology)

该项目有两个目标：(1)研究人员通过在宿主实验室的工作从哺乳动物细胞中分离出一个新的转化生长因子-β超家族成员来学习分子克隆，学习最先进的膜蛋白克隆；(2)从胚胎中建立海胆cDNA文库，并利用这些文库和宿主实验室的资源，寻找转化生长因子-β受体和整合素受体的克隆。研究人员将首先使用低严格杂交来寻找转化生长因子-β和整合素受体的海胆克隆。如果发现太多的克隆，她将设计冗余的寡核苷酸探针，并使用聚合酶链式反应技术试图从她的文库中分离出海胆探针，或者准备针对整合素受体中冗余的肽序列的抗体，并筛选表达库中的克隆。最后，她将使用自己针对可能的200 kDa层粘连蛋白受体的抗体来筛选海胆文库中的克隆。长期目标是了解细胞表面的受体分子如何与细胞外基质的特定分子相互作用，以信号或帮助细胞运动和胚胎的形态发生。有初步证据表明，细胞外基质蛋白，层粘连蛋白，通过与细胞受体的相互作用，可以刺激或中介这些胚胎的原肠形成，研究人员已经开始从海胆胚胎中提纯与细胞外基质成分结合的膜蛋白。她发现了一种结合层粘连蛋白的蛋白质--首次在海胆胚胎中发现了一种基质受体，目前正在使用从海胆到哺乳动物的抗体进化树寻找可能结合层粘连蛋白或纤维连接蛋白的其他整合素蛋白质，这些蛋白质显著重叠。这位研究人员是棘皮动物卵子和胚胎翻译调控方面的专家，她正在改变自己实验室的重点，研究膜受体和细胞外基质分子在海胆胚胎形态发生运动中的作用。海胆是一种易于可视化和操作的发育模型，它的研究对于进一步理解细胞-细胞外基质相互作用作为胚胎细胞运动和分化的决定因素，特别是原肠形成的关键过程，以及类似的过程，如果蝇的成像盘形成或脊椎动物的神经形成具有重要意义。该项目进一步推动了VPW项目的目标，为女性提供通过研究提升她们在科学或工程领域的职业生涯的机会，并通过调查员作为东道主校园榜样的提高知名度，鼓励其他女性在这些领域追求职业生涯。促进第二个目标的拟议活动包括：举办讲座/研讨会；在一些研究生和本科生课程中讲课，包括发育生物学、生物学入门和高级细胞生物学；与参加正在进行的生物学午餐的研究生妇女合作；并在细胞生物学女性协会(AWIS)的剑桥分会上发表演讲。

The project has two objectives: (1) for the investigator to learn molecular cloning by isolating a new member of the TGF- beta superfamily from mammalian cells through work in the host laboratory, learning state-of-the-art cloning of membrane proteins; and (2) to make sea urchin cDNA libraries from embryos, and with these and host laboratory resources, to search for clones for TFG- beta receptors and integrin receptors. The investigator will begin by using low-stringency hybridization to look for sea urchin clones of TGF-beta and integrin receptors. If too many clones are found she will design redundant oligo-nucleotide probes and use PCR techniques to try to isolate sea urchin probes from her libraries, or prepare antibodies to redundant peptide sequences in the integrin receptors and screen expression libraries for clones. Finally, she will use her own antibodies to the putative 200kDa-laminin receptor to screen for clones in the sea urchin libraries. The long term goal is to understand how receptor molecules on the surface of cells interact with specific molecules of the extracellular matrix to signal or aid cell movements and morphogenesis of embryos. With preliminary evidence that the interaction of the extracellular matrix protein, laminin, by interacting with cell receptors may stimulate or mediate gastrulation these embryos, the investigator has begun to purify membrane proteins from sea urchin embryos that bind to extracellular matrix components. She has found one protein that binds laminin--the first identification of a matrix receptor in sea urchin embryos, and is currently searching for other integrin proteins that may bind laminin or fibronectin using an antibody evolutionary tree from sea urchins to mammals, significantly overlap. The investigator, an expert on the regulation of translation in echinoderm egg and embryos, is changing the emphasis of her own laboratory to study the role of membrane receptors and extracellular matrix molecules in morphogenetic movements of sea urchin embryos. The studies on sea urchins, a model for the easy visualization and manipulation of development, are potentially very important for furthering our understanding of cell-extracellular matrix interactions as determinants of embryonic cell movement and differentiation, particularly the critical processes of gastrulation, and similar processes such as imaginal disc formation in Drosophila or neurulation in vertebrates. This project furthers VPW program objectives to provide opportunities for women to advance their careers in science or engineering through research, and to encourage other women to pursue careers in these areas through the investigator's enhanced visibility as a role model on the host campus. The proposed activities which contribute to the second objective include: giving lectures/seminars; lecturing in a number of graduate and undergraduate courses, including developmental biology, introductory biology, and advanced cell biology; working with graduate student women participating in the on-going biology brown-bag lunches; and giving a lecture to the Cambridge Chapter of the Association of Women in Cell Biology (AWIS).