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

该项目有两个目标：(1)通过在宿主实验室的工作，从哺乳动物细胞中分离TGF- β超家族新成员，学习分子克隆，学习膜蛋白的最新克隆技术；(2)利用胚胎和宿主实验室资源构建海胆cDNA文库，寻找TFG- β受体和整合素受体的克隆。研究人员将开始使用低强度杂交寻找tgf - 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PCR技术试图从她的文库中分离海胆探针，或者准备针对整合素受体中冗余肽序列的抗体，并筛选克隆的表达文库。最后，她将使用她自己的抗体对假定的200kda -层粘连蛋白受体进行筛选，在海胆文库中克隆。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，以指示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与细胞受体的相互作用可能刺激或介导这些胚胎的原肠胚形成，研究者已经开始从海胆胚胎中纯化与细胞外基质成分结合的膜蛋白。她已经发现了一种结合层粘连蛋白的蛋白质——这是第一次在海胆胚胎中发现基质受体。目前，她正在利用从海胆到哺乳动物的抗体进化树，寻找其他可能结合层粘连蛋白或纤维连接蛋白的整合蛋白。作为棘皮动物卵子和胚胎翻译调控方面的专家，她正在将自己实验室的重点转向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。对海胆的研究是一种容易可视化和操纵发育的模型，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，特别是原肠胚形成的关键过程，可能非常重要。以及类似的过程，如果蝇的想象盘形成或脊椎动物的神经形成。该项目进一步推进了VPW项目的目标，即通过研究为女性提供在科学或工程领域发展的机会，并通过研究者在主办校园中作为榜样的知名度提高，鼓励其他女性在这些领域追求职业。有助于实现第二个目标的拟议活动包括：举办讲座/研讨会；在许多研究生和本科生课程中授课，包括发育生物学、生物学入门和高级细胞生物学；与研究生女学生一起参加正在进行的生物棕袋午餐；并在细胞生物学女性协会（AWIS）的剑桥分会发表演讲。

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).