Functional of an Unconventional Myosin in Tetrahymena

智力上的功绩。通过吞噬作用使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。内化的颗粒包含在被称为吞噬小体的细胞内可移动的小泡中。吞噬小体与溶酶体的融合为内化颗粒的消化提供了化学环境。在四膜虫中，吞噬小体似乎是垂直排列的，当吞噬小体向细胞的后端移动时，这种排列保持不变。MYO1是一种含有MyTH4/FERM结构域的XIV肌球蛋白，在敲除MYO1后，吞噬小体的运动要么停止，要么变得随机。抑制肌动蛋白细丝组装诱导野生型细胞中吞噬小体的随机运动。因此，Myo1和肌动蛋白细丝都是维持吞噬小体运动方向性所必需的。从Myo1融合到FERM结构域的GFP定位于吞噬小体，吞噬小体似乎附着在细胞骨架基本体的纵行上。本项目旨在研究吞噬小体运动的方向性。该项目将利用四膜虫中吞噬小体的运动性来检验这一假说，即吞噬小体的定向运动涉及Myo1费姆结构域与基底体相关肌动蛋白细丝的相互作用，后者是吞噬小体运动的假定轨迹。这一假说将在体内和体外进行测试。为了分析体内吞噬小体的运动性，将用荧光小球作为吞噬小体的标记来挑战GFP-FERM菌株。延时共聚焦显微镜将被用来监测吞噬小体运动的速度和方向，这与细胞骨架基底的纵向排列有关。预计过表达的FERM将与内源性Myo1竞争结合位点，干扰吞噬小体运动的方向性。该假说的一个预测是，细胞骨架基本体纵向排列的中断将导致吞噬小体的随机运动。为了验证这一预测，将使用热休克来导致基底体排列不连续。在热休克细胞恢复正常吞噬后，将分析吞噬小体的运动。为了进一步证明吞噬小体沿着纵向排列的基本小体移动，细胞骨架将从GFP-FERM菌株中分离出来。将用荧光珠攻击的细胞的非变性总细胞裂解物添加到细胞骨架部分，并分析吞噬小体的运动。这个项目的结果将大大有助于我们理解吞噬体如何保持方向性。广泛的影响一项教育计划为本科生提供研究培训，并为中学生提供科学研讨会。在PI的实验室里，研究生们学到的不仅仅是如何在实验中使用一项专门的技术。他们是研究经验的所有阶段的参与者，包括实验设计、数据获取、数据解释和准备发表的数据。研究生被要求参加两周一次的以本科生研究报告为特色的系级研讨会。

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.