Collaborative Research: Intracellular Patterning in the Ciliate Cell Cortex

本项目旨在利用单细胞原生动物嗜热四膜虫确定在细胞表面组织特殊结构的普遍机制。研究的结构包括对进食和排泄都很重要的结构。关于如何将新合成的细胞表面成分以适当的数量运送到适当的位置的知识将会产生。该项目将通过独立和基于课程的研究经验相结合，吸引早期职业科学家（主要是本科生）从事遗传学，细胞生物学和高级显微镜。这项工作试点了一个项目，招募来自代表性不足背景的学生进入有指导的合作研究团队，并长期承诺学徒。在此过程中，我们将开发“可见细胞”，这是一个综合的教学和研究资源，包括在工程细胞系中表达的荧光细胞探针，并提供给更广泛的研究界。该集合将包括表达荧光标记蛋白质的细胞，这些蛋白质可以在实验室和教室中研究特定的细胞器，膜系统和细胞骨架成分。本研究的重点是拓宽皮质结构域（BCD）1基因，当该基因失活时，会导致多个口的组装和多余的排泄孔。该基因的序列分析表明其产物类似于参与膜运输的蛋白质。实验旨在验证Bcd1蛋白通过局部激酶激活促进内吞作用（回收多余的表面物质）的假设。这些实验包括对膜运输和细胞信号通路的药理学和遗传操作，以阐明这些通路的稳态如何导致细胞表面细胞器的适当组装。最后，我们将鉴定和表征导致janusA表型的基因，这是整个腹侧细胞器模式的镜像复制。总之，这些对BCD1和janusA的研究将提供对决定主要细胞表面结构和整体细胞模式的定位和尺寸的机制的理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project seeks to identify universal mechanisms that organize specialized structures on the cell surface using the single-celled protozoan Tetrahymena thermophila. Structures studied include ones important for both feeding and excretion. Knowledge will be generated as to how newly synthesized cell-surface components are delivered to the proper location and in the proper abundance. This project will engage early career scientists (primarily undergraduates) in genetics, cell biology, and advanced microscopy through a combination of independent and course-based research experiences. This effort pilots a program to recruit students from under-represented backgrounds into mentored, collaborative research teams with long-term committed apprenticeships. Along the way, we will develop the “Visible Cell”, a comprehensive teaching and research resource consisting of fluorescent cellular probes expressed in engineered cell-lines and made available to the broader research community. This collection will include cells that express fluorescently-tagged proteins spotlighting specific organelles, membrane systems, and cytoskeletal components which can be studied in both the laboratory and the classroom. This research focuses on the Broadened Cortical Domain (BCD)1 gene, which when disabled, results in the assembly of multiple mouths and supernumerary excretory pores. Sequence analysis of this gene reveals that its product resembles a protein involved in membrane trafficking. Experiments are designed to test the hypothesis that the Bcd1 protein promotes endocytosis (retrieval of excess surface materials) through localized kinase activation. These experiments include pharmacological and genetic manipulation of membrane trafficking and cell signaling pathways to elucidate how homeostasis of these pathways leads to proper assembly of cell surface organelles. Finally, we will identify and characterize the gene that causes the janusA phenotype, a mirror-image duplication of the entire ventral organelle pattern. Together, these studies of BCD1 and janusA will provide understanding of mechanisms that determine both the localization and dimensions of major cell surface structures and global cell patterning.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.