CAREER: Single-molecule dissection of the Partitioning Defective cell polarity machinery

这个项目的目标是更好地理解细胞如何建立和维持一种称为细胞极性的内部“方向感”。我们体内的细胞依靠这种固有的极性在组织中定位自己。例如，为了正常运作，我们肠道中的细胞需要知道细胞的哪一边面对管腔空间，食物在哪里被消化，哪一边面对潜在的血管，哪里需要吸收营养。这个项目使用新的、高度敏感的技术来研究细胞内的分子如何相互作用来产生使细胞极化的空间模式。为了确保结果是可推广的，将并行研究来自蠕虫和老鼠的极化细胞类型。这一结果有望阐明动物细胞如何在发育中的胚胎和成年身体内定位。这个项目所需的大部分工作将由从德克萨斯大学奥斯汀分校的不同学生群体中招募的本科生研究人员进行。一年级本科生研究人员将学习基因组编辑技术，同时进行大规模搜索新的极性基因，作为基于课程的本科生研究体验的一部分。这些学生中的一部分将以学长的身份更深入地参与研究，在首席科学家的实验室内进行独立的项目。因此，该项目将有助于培养和发展一支多样化的STEM工作队伍。细胞极性是分子和活动在细胞内的不对称定位，对动物组织的发育和动态平衡至关重要。支架蛋白PAR-3是动物和许多不同极化细胞类型中极性的关键调节因子。本课题组此前已发现，在秀丽线虫受精卵中，PAR-3形成含有中心极性激酶aPKC的寡聚复合体，这些复合体通过肌动蛋白皮质流被带到受精卵的前面。本项目的目标是确定1)PAR-3复合体在线虫受精卵中是如何组装和分解的，以及2)这些机制在哺乳动物细胞中是否保守。为了解决这些问题，该小组已经开发并将应用一种创新的单细胞、单分子生物化学方法，允许直接从极化细胞中提取和量化蛋白质复合体。通过测量线虫受精卵中PAR-3复合体的组成和动力学，该项目将阐明PAR-3复合体组装的生化基础。这些实验将包括已知的参与者和从蛋白质组筛选中识别出的新分子。然后，这一方法将扩展到小鼠胚胎干细胞，这是一种哺乳动物上皮极性模型，将使这些发现能够在动物系统中推广。总之，这项工作将阐明低聚PAR-3复合体是如何组装和控制的，以便在活体中实现适当的细胞极化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The goal of this project is to better understand how cells establish and maintain an internal “sense of direction” called cell polarity. The cells in our bodies rely on this intrinsic polarity to orient themselves within a tissue. For example, to function properly, cells in our intestines need to know which side of the cell is facing the luminal space, where food is being digested, and which side is facing the underlying blood vessels, where nutrients need to be absorbed. This project uses new, highly sensitive techniques to study how molecules inside cells can interact to produce spatial patterns that polarize a cell. To ensure that the results are generalizable, polarizing cell types from both worms and mice will be studied in parallel. The results are expected to shed light on how animal cells orient themselves within a developing embryo and in the adult body. Much of the work required for this project will be carried out by undergraduate researchers recruited from the diverse student body at the University of Texas at Austin. Freshman undergraduate researchers will learn genome editing technologies while conducting a large-scale search for new polarity genes, as part of a course-based undergraduate research experience. A subset of these students will participate more deeply in the research as upperclassmen, by taking on independent projects within the lead scientist’s lab. As a result, this project will contribute to training and developing a diverse STEM workforce.Cell polarity is the asymmetric localization of molecules and activities within a cell, and is essential for the development and homeostasis of animal tissues. The scaffold protein PAR-3 is a key regulator of polarity across animals and in many different polarized cell types. This research group has previously shown that in Caenorhabditis elegans zygotes, PAR-3 forms oligomeric complexes containing the central polarity kinase aPKC, and these complexes are carried to the anterior of the zygote by actomyosin cortical flows. The goals of the present project are to determine 1) how PAR-3 complexes are assembled and disassembled in C. elegans zygotes, and 2) whether these mechanisms are conserved in mammalian cells. To address these questions, the group has developed and will apply an innovative single-cell, single-molecule biochemistry approach that allows direct extraction and quantification of protein complexes from polarizing cells. By measuring the composition and dynamics of PAR-3 complexes in polarizing C. elegans zygotes, the project will shed light on the biochemical basis for PAR-3 complex assembly. These experiments will incorporate both known players and new molecules identified from proteomic screens. This approach will then be extended to mouse embryonic stem cells, a mammalian epithelial polarity model that will enable the findings to be generalized across animal systems. Together, this work will elucidate how oligomeric PAR-3 complexes are assembled and controlled to enable proper cell polarization in vivo.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.