Functional of an Unconventional Myosin in Tetrahymena

知识价值。通过吞噬作用将微粒内化对多种细胞类型至关重要，包括免疫系统的专业吞噬细胞和利用这一过程捕获食物的微生物。内化颗粒包含在称为吞噬体的细胞内运动囊泡中。吞噬体与溶酶体的融合为消化内化颗粒提供了化学环境。在四膜虫中，吞噬体看起来是垂直排列的，当吞噬体向细胞的后端移动时，这种排列保持不变。MYO1（一种含有MyTH4/FERM结构域的XIV类肌球蛋白）敲除后，吞噬体的运动停止或变得随机。抑制肌动蛋白丝组装可诱导野生型细胞吞噬体的随机运动。因此，Myo1和肌动蛋白丝都是维持吞噬体运动的方向性所必需的。与Myo1的FERM结构域融合的GFP定位于吞噬体，吞噬体似乎附着在细胞骨架基底的纵行上。本项目旨在研究吞噬体运动的方向性。该项目将利用四膜虫中吞噬体的运动来验证一个假设，即吞噬体的定向运动涉及Myo1 FERM结构域与基础体相关肌动蛋白丝的相互作用，后者是吞噬体运动的假定路径。这一假设将在体内和体外进行验证。为了分析吞噬体在体内的运动，GFP-FERM菌株将用荧光珠作为吞噬体的标记。延时共聚焦显微镜将用于监测吞噬体运动的速度和方向与细胞骨架基底体的纵向排列有关。预计过表达的FERM将与内源性Myo1竞争结合位点，并干扰吞噬体运动的方向性。该假说的一个预测是，细胞骨架基底纵向排列的破坏将诱导吞噬体的随机运动。为了验证这一预测，将使用热冲击来诱导基底体排列中的不连续性。在热休克细胞恢复正常吞噬后，分析吞噬体的运动。为了进一步证明吞噬体沿着基底体的纵向行导航，将从GFP-FERM菌株中分离出细胞骨架。用荧光珠攻击的细胞未变性的总细胞裂解液将加入到细胞骨架部分，并分析吞噬体的运动。这个项目的结果将显著有助于我们理解吞噬体如何保持方向性。更广泛的影响一项教育计划为本科生提供研究培训，为中学生提供科学讲习班。在PI实验室的研究学生学到的不仅仅是如何在实验中使用专门的技术。他们参与研究经历的所有阶段，包括实验设计、数据获取、数据解释和准备发表数据。研究生必须每两周参加一次以本科生研究报告为特色的部门研讨会。

Intellectual Merit. Internalization of particulates through phagocytosis is of fundamental importance to diverse cell types including professional phagocytes of the immune system and microorganisms that use the process for capturing food. Internalized particulates are contained within motile intracellular vesicles known as phagosomes. Fusion of phagosomes with lysosomes provides the chemical environment for digestion of the internalized particulates. In Tetrahymena, phagosomes appear to be vertically aligned, and the alignment is maintained as phagosomes move toward the posterior end of the cell. Motility of phagosomes either ceases or becomes random in a knockout of MYO1, a class XIV myosin that contains MyTH4/FERM domains. Inhibition of actin filament assembly induces random motility of phagosomes in wild type cells. Therefore, both Myo1 and actin filaments are required for maintaining directionality of phagosome motility. GFP fused to the FERM domain from Myo1 localizes to phagosomes, which appear to be attached to the longitudinal rows of cytoskeleton basal bodies. This project aims to investigate directionality of phagosome motility. The project will use motility of phagosomes in Tetrahymena to test the hypothesis that directed motility of phagosomes involves the interaction of the Myo1 FERM domain with basal body-associated actin filaments that are putative tracks for phagosome motility. The hypothesis will be tested in vivo and in vitro. For analysis of phagosome motility in vivo, the GFP-FERM strain will be challenged with fluorescent beads as markers for phagosomes. Time-lapse confocal microscopy will be used to monitor the velocity and direction of phagosome motility in relation to the longitudinal alignment of cytoskeleton basal bodies. It is expected that overexpressed FERM will compete with endogenous Myo1 for binding sites and interfere with directionality of phagosome motility. A prediction of the hypothesis is that disruptions in the longitudinal alignment of cytoskeleton basal bodies will induce random motility of phagosomes. To test this prediction, heat shock will be used to induce discontinuities in the alignment of basal bodies. Phagosome motility will be analyzed in the heat-shocked cells after they have resumed normal phagocytosis. To further demonstrate that phagosomes navigate along longitudinal rows of basal bodies, cytoskeletons will be isolated from the GFP-FERM strain. Nondenatured total cell lysate from cells challenged with fluorescent beads will be added to the cytoskeleton fraction, and phagosome motility will be analyzed. The outcomes of this project will significantly contribute to our understanding of how phagosomes maintain directionality.Broader ImpactsAn education plan provides research training for undergraduates and science workshops for middle school students. Research students in the PI's laboratory learn more than how to use a specialized technique in an experiment. They are participants in all phases of the research experience including experimental design, data acquisition, data interpretation, and preparation of data for publication. Research students are required to attend biweekly departmental seminars that feature undergraduate research presentations.