Collaborative Research: Molecular mechanisms governing the cytoskeleton-mediated motility and distribution of peroxisomes and mitochondria in plants

本研究旨在解决细胞生物学中与植物（包括作物）生长和繁殖直接相关的一个基本问题。植物细胞动员其高度动态的亚细胞区室，即细胞器，来完成生长发育的生理功能。该项目将揭示细胞器在响应内部和外部信号时沿着称为肌动蛋白丝的分子轨道移动和分布的鲜为人知的机制。重点将放在过氧化物酶体和线粒体上，这两种细胞器在物理和生物化学上相互联系，对植物的能量代谢和生存至关重要。负责这两种细胞器运动和分布的分子机制将被解剖，使用小芥菜植物拟南芥。所获得的知识将为与植物细胞器运动相关的原理提供基本见解，从而为这些分子机制的进化提供视角。此外，该项目还将培养研究生和本科生的现代生物学技能。在课堂上为一年级本科生提供实践研究经验，目的是让他们留在科学专业。为分子生物学、遗传学、细胞生物学、生理学和计算生物学的本科生提供以发现为基础的暑期培训，为他们毕业后从事科学事业或接受高级培训做好准备。细胞骨架依赖的细胞器的主动运动和分布对细胞生长、分裂和信号传导具有重要意义。然而，细胞器如何招募细胞骨架运动蛋白以沿着细胞骨架轨道进行定向运输是复杂的，并且经常存在争议。该项目主要研究植物过氧化物酶体和线粒体的运动和分布的分子机制，这些细胞器是能量代谢和植物生存所必需的多功能和代谢相关细胞器。在拟南芥中，一项探索性的工作检测了过氧化物酶体沿着肌动蛋白丝的定向运输，并发现了在过氧化物酶体/线粒体和细胞骨架之间的界面上起作用的候选蛋白，用于细胞器的运动和分布。据推测，过氧化物酶体/线粒体沿着肌动蛋白丝的主动长距离运动是由细胞器特异性受体/接头蛋白和gtpase调节的，gtpase募集肌动蛋白相关的Myosin XI马达，为运动提供能量。该项目包括三个目标：(1)确定肌动蛋白结合蛋白过氧化物酶体和线粒体分裂1 （PMD1）在介导过氧化物酶体/线粒体与肌动蛋白丝的捆绑中的作用；(2)通过检测tonsoku相关蛋白1 （TSA1）、RABE1c GTPase和过氧化物酶PEX5的作用，解剖将肌球蛋白XI招募到过氧化物酶体的接头复合体；(3)通过检测心肌病相关蛋白（CMYA）和MIRO1 GTPase的功能，解剖将肌球蛋白XI招募到线粒体的接头复合体。蛋白质免疫亲和纯化、蛋白质组学、活细胞成像、遗传学和生理表型将被用于理清调节细胞器运动的过氧化物酶体/线粒体和肌动蛋白细胞骨架之间的相互作用，并促进对细胞器运动对植物适应性影响的理解。此外，该项目还将对两所大学的研究生和本科生进行现代生物和计算技术方面的培训。在课堂上为一年级本科生提供实践研究经验，目的是让他们留在科学专业。为分子生物学、遗传学、细胞生物学、生理学和计算生物学的本科生提供以发现为基础的暑期培训，为他们毕业后从事科学事业或接受高级培训做好准备。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 research is aimed at addressing a fundamental question in cell biology that is directly associated with growth and reproduction of plants, including crops. Plant cells mobilize their highly dynamic subcellular compartments, known as organelles, to fulfill physiological functions in growth and development. This project will uncover the poorly understood mechanisms by which organelles move and distribute along molecular tracks called actin filaments within plant cells in response to internal and external signals. The focus will be on peroxisomes and mitochondria, two organelles that are physically and biochemically connected and essential for energy metabolism and survival of plants. The molecular machinery responsible for the motility and distribution of these two types of organelles will be dissected, using the small mustard plant Arabidopsis. Knowledge gained will provide fundamental insights into the principles associated with the motility of plant organelles, thereby giving perspectives on how these molecular machineries evolved. In addition, the project will train graduate and undergraduate students in modern biological skills. Hands-on research experience will be provided to first-year undergraduate students in the classroom with the goal of retaining them in science majors. Discovery-based summer trainings will be provided to undergraduates in molecular biology, genetics, cell biology, physiology and computational biology in order to prepare them for a scientific career or advanced training after graduation. Cytoskeleton-dependent active movement and distribution of organelles are fundamentally significant for cell growth, division, and signaling. However, how organelles recruit cytoskeletal motor proteins for their directional transport along the cytoskeletal tracks is complex and often under debate. This project focuses on molecular mechanisms underlying the motility and distribution of plant peroxisomes and mitochondria, multifunctional and metabolically linked organelles essential for energy metabolism and plant viability. An exploratory effort detected directional transport of peroxisomes along actin filaments and discovered candidate proteins functioning at the interface between peroxisomes/mitochondria and the cytoskeleton for organellar motility and distribution in Arabidopsis thaliana. It is hypothesized that the active long-distance movement of peroxisomes/mitochondria along actin filaments is regulated by organelle-specific receptor/adaptor proteins and GTPases, which recruit the actin-associated Myosin XI motors that energize the motility. The project includes three aims: (1) Determining the role of the actin-binding protein Peroxisomal and Mitochondrial Division 1 (PMD1) in mediating the tethering of peroxisomes/mitochondria to the actin filaments; (2) Dissecting the adaptor complex that recruits Myosin XI to the peroxisome by testing the roles of TONSOKU-associated protein 1 (TSA1), the RABE1c GTPase, and the peroxin PEX5; and (3) Dissecting the adaptor complex that recruits Myosin XI to mitochondria by testing the functions of Cardiomyopathy-Associated Protein (CMYA) and the MIRO1 GTPase. Protein immuno-affinity purification, proteomics, live-cell imaging, genetics and physiological phenotyping will be employed to untangle interactions between peroxisomes/mitochondria and the actin cytoskeleton that regulate organelle motility and advance the understanding of the impact of organelle motility on plant fitness. In addition, the project will train both graduate and undergraduate students in modern biological and computational techniques at both universities. Hands-on research experience will be provided to first-year undergraduate students in the classroom with the goal of retaining them in science majors. Discovery-based summer trainings will be provided to undergraduates in molecular biology, genetics, cell biology, physiology, and computational biology in order to prepare them for a scientific career or advanced training after graduation.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.