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

这项研究旨在解决细胞生物学中的一个基本问题，该问题与植物（包括农作物）的生长和繁殖直接相关。 植物细胞调动其高度动态的亚细胞区室，称为细胞器，以实现生长和发育中的生理功能。 这个项目将揭示知之甚少的机制，细胞器移动和分布沿着分子轨道称为肌动蛋白丝在植物细胞内响应内部和外部信号。 重点将放在过氧化物酶体和线粒体，这两个细胞器是物理和生化连接，对能量代谢和植物生存至关重要。 负责这两种类型的细胞器的运动和分布的分子机制将被解剖，使用小芥菜植物拟南芥。 所获得的知识将提供与植物细胞器的运动性相关的原理的基本见解，从而对这些分子机器如何进化提供观点。 此外，该项目还将对研究生和本科生进行现代生物技能培训。 实践研究经验将提供给一年级的本科生在课堂上与保留他们在科学专业的目标。 将为分子生物学、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，以便为他们毕业后的科学生涯或高级培训做好准备。 细胞器依赖于细胞激素的主动运动和分布对于细胞生长、分裂和信号传导具有根本意义。 然而，细胞器如何招募细胞骨架马达蛋白，使其沿着细胞骨架轨道定向运输是复杂的，经常处于争论之中。 该项目的重点是植物过氧化物酶体和线粒体的运动和分布的分子机制，多功能和代谢相关的细胞器对能量代谢和植物生存力至关重要。 一项探索性的工作检测到过氧化物酶体沿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质，用于拟南芥中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受细胞器特异性受体/衔接蛋白和GTP酶的调节，其募集肌动蛋白相关的肌球蛋白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第1区（PMD 1）在介导过氧化物酶体/线粒体与肌动蛋白丝的束缚中的作用;（2）通过测试TONSOKU相关蛋白1（TSA 1）、RABE 1c GTPase和过氧化物酶PX 5的作用，剖析将肌球蛋白Xi招募到过氧化物酶体的衔接复合物;（3）通过检测心肌病相关蛋白（CMYA）和MIRO 1 GTdR的功能，分析将肌球蛋白Xi募集到线粒体的接头复合物。 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来解开过氧化物酶体/线粒体和肌动蛋白细胞骨架之间的相互作用，调节细胞器运动和推进细胞器运动对植物健身的影响的理解。 此外，该项目还将在两所大学对研究生和本科生进行现代生物和计算技术方面的培训。 实践研究经验将提供给一年级的本科生在课堂上与保留他们在科学专业的目标。 以发现为基础的暑期培训将提供给分子生物学、遗传学、细胞生物学、生理学和计算生物学的本科生，以便他们为毕业后的科学生涯或高级培训做好准备。该奖项反映了NSF的法定使命，并通过使用基金会的智力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.