Intracellular cargo transport in Toxoplasma gondii

项目总结 细胞内货物转运是所有真核细胞中普遍存在的重要细胞过程。然而， 货物运输的机制只在少数“模式”物种中得到了广泛的研究， 在我们对其他真核生物组的货物运输的理解上留下了很大的知识空白。我的实验室 最近在Apicomplexan原生动物寄生虫中发现了一种新的货物运输机制 弓形虫；阐明这种细胞内转运系统的机制细节是重点 关于这项提议。弓形虫是弓形虫门的一部分，该门含有大量的药用 重要的寄生虫包括疟疾的病原体疟原虫和隐孢子虫。那 会导致危及生命的腹泻病。因此，除了提供对不同机制的洞察之外， 在进化分化的真核生物中存在的货物运输的研究，了解货物的分子基础 在顶端复合体中的运输可能导致识别潜在的抗寄生虫药物开发靶点。 我们之前发表的初步数据表明，两种细胞骨架蛋白，肌动蛋白和一个 非常规肌球蛋白(MyoF)是运输多种细胞内货物所必需的。虽然 MyoF与特征良好的货物转运蛋白Myosin V在结构上具有相似性，我们证明了MyoF 不直接与其膜结合的货物相关联。相反，MyoF是底层肌动蛋白的组织者 细胞骨架。为了全面了解这种货物运输机制，我们的目标是确定 货物运输所需的其他分子参与者，并定义这些 蛋白质与MyoF和肌动蛋白共同驱动货物移动。具体地说，我们将回答以下问题 悬而未决的问题：MyoF的活动是如何调节的？泡状物是如何与肌动蛋白相联系的 细胞骨架？是否有其他蛋白质与MyoF合作来控制组织和动态 肌动蛋白细胞骨架的？为了解决这些问题，我们将利用三个截然不同但互为补充的 实验方法：(1)寄生虫遗传学和细胞生物学；(2)活细胞成像和定量囊泡 示踪法和(3)体外生物物理方法。我的实验室在十字路口占据了一个独特的利基位置 在寄生虫学和分子马达领域之间。利用这些跨学科的方法使我们成为理想的 为这一未被研究的过程提供新的见解。

PROJECT SUMMARY Intracellular cargo transport is a vital and ubiquitous cellular process that occurs in all eukaryotic cells. However, the mechanisms of cargo transport have only been extensively investigated in a small number of “model” species, leaving a large knowledge gap in our understanding of cargo transport in other eukaryotic groups. My laboratory has recently uncovered a novel mechanism of cargo transport in the Apicomplexan protozoan parasite Toxoplasma gondii; elucidating the mechanistic details underlying this intracellular transport system is the focus on this proposal. T. gondii is part of the phylum Apicomplexan, which contains a large number of medically important parasites including Plasmodium spp., the causative agent of malaria and Cryptosporidium spp. that can cause life-threatening diarrheal disease. Thus, in addition to providing insight into the diverse mechanisms of cargo transport that exist in evolutionary divergent eukaryotes, understanding the molecular basis of cargo transport in Apicomplexa could lead to the identification of potential targets of anti-parasitic drug development. Our previously published and preliminary data demonstrate that two cytoskeletal proteins, actin and an unconventional myosin (MyoF) are required for the movement of a wide range of intracellular cargo. Although MyoF has structural similarity to the well-characterized cargo transporter myosin V, we demonstrate that MyoF does not associate directly with its membrane bound cargo. Instead, MyoF is an organizer of the underlying actin cytoskeleton. In order to gain a complete picture of this cargo transport mechanism, our goal is to identify additional molecular players that are required for cargo transport and to define the mechanisms by which these proteins drive cargo movement in collaboration with MyoF and actin. Specifically, we will answer the following outstanding questions: How is the activity of MyoF regulated? How does vesicular cargo associate with the actin cytoskeleton? Are there additional proteins that cooperate with MyoF to control the organization and dynamics of the actin cytoskeleton? To address these questions, we will utilize three distinct, yet complementary, experimental approaches: (1) parasite genetics and cell biology, (2) live cell imaging and quantitative vesicle tracking and (3) in vitro biophysical approaches. My laboratory occupies a unique niche at the intersection between parasitology and molecular motors fields. Utilizing these interdisciplinary approaches makes us ideally positioned to provide new insights into this understudied process.