Functional of an Unconventional Myosin in Tetrahymena

智力优势。通过吞噬作用内化颗粒对于不同的细胞类型具有根本重要性，包括免疫系统的专职吞噬细胞和使用该过程捕获食物的微生物。 内化的颗粒包含在称为吞噬体的能动细胞内囊泡中。 吞噬体与溶酶体的融合提供了消化内化颗粒的化学环境。 在四膜虫中，吞噬体似乎是垂直排列的，当吞噬体向细胞的后端移动时，这种排列得以维持。 在MYO 1（一种含有MyTH 4/FERM结构域的XIV类肌球蛋白）敲除后，吞噬体的运动性停止或变得随机。 肌动蛋白丝组装的抑制诱导吞噬体在野生型细胞中的随机运动。 因此，Myo 1和肌动蛋白丝都需要维持吞噬体运动的方向性。 GFP融合到来自Myo 1的FERM结构域定位于吞噬体，其似乎附着于细胞骨架基体的纵行。 本研究旨在探讨吞噬体运动的方向性。 该项目将使用四膜虫中的吞噬体的运动性来测试以下假设：吞噬体的定向运动性涉及Myo 1 FERM结构域与基体相关的肌动蛋白丝的相互作用，所述肌动蛋白丝是吞噬体运动性的推定轨道。 该假设将在体内和体外进行检验。 为了分析体内吞噬体运动性，GFP-FERM菌株将用荧光珠作为吞噬体的标记物进行攻击。 延时共聚焦显微镜将用于监测与细胞骨架基体纵向排列相关的吞噬体运动的速度和方向。 预计过表达的FERM将与内源性Myo 1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，细胞骨架基体的纵向排列的中断将诱导吞噬体的随机运动。 为了检验这一预测，将使用热冲击来诱导基体排列的不连续性。 在热休克细胞恢复正常吞噬作用后，将在热休克细胞中分析吞噬体运动性。 为了进一步证明吞噬体沿沿着纵向行的基体导航，将从GFP-FERM菌株中分离细胞骨架。 将来自用荧光珠激发的细胞的非变性总细胞裂解物加入细胞骨架组分中，并分析吞噬体运动性。 这个项目的成果将大大有助于我们理解吞噬体如何保持方向性。更广泛的影响一个教育计划提供了研究培训的本科生和科学讲习班的中学生。 PI实验室的研究生学习的不仅仅是如何在实验中使用专业技术。 他们是研究经验的所有阶段的参与者，包括实验设计，数据采集，数据解释和数据准备出版。 研究生必须参加每两周一次的部门研讨会，其中包括本科生的研究报告。

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.