Using a Viral Protein to Study the Functional Organization of ER-mitochondrial Membrane Contacts and Trafficking Across this Compartment

内质网（ER）和线粒体之间的膜接触，被称为线粒体相关膜（MAM），是在多种细胞功能中起关键作用的通讯枢纽，包括内质网到线粒体钙（Ca2+）信号，线粒体生物能量学，脂质交换，先天免疫和细胞凋亡。MAM现在正在成为内质网到线粒体运输细胞蛋白和病毒蛋白的一个位点。该项目描述了一种巨细胞病毒（CMV）蛋白，称为UL37外显子1蛋白（pUL37x1），其靶向MAM，改变其某些功能，并从内质网转运到线粒体。该项目将研究蛋白质如何使用MAM从内质网到线粒体的交通。预计CMV pUL37x1转运使用MAM囊泡或系链介导的机制运输到线粒体。该项目将使用互补的抑制方法（sirna、敲除细胞、显性阴性突变体和化学抑制剂）和MAM栓系物或MAM囊泡途径成分的过表达来测试MAM栓系物或囊泡成分是否构成ER对线粒体运输的基础。更广泛的影响这些研究将影响细胞内蛋白质运输领域，以及教育和社会（技术发展）。该项目旨在了解MAM组织及其调控，影响细胞生理和反应。使用多焦点结构照明显微镜（MSIM）将推进细胞内蛋白质运输领域，更广泛地说，细胞成像。这些研究将包括对本科生研究人员的培训，特别是将继续支持参与的实验室在培训科学领域代表性不足的少数民族方面的努力。由于本项目采用多学科方法，学生将受益于共聚焦活细胞成像、MSIM成像、分子病毒学和生化方法的培训，参与科学讨论，并在科学会议上展示他们的工作。

INTELLECTUAL MERITMembrane contacts between the endoplasmic reticulum (ER) and mitochondria, known as mitochondria-associated membranes (MAM) are communication hubs that play crucial roles in diverse cellular functions, including ER to mitochondrial calcium (Ca2+) signaling, mitochondrial bioenergetics, lipid exchange, innate immunity, and apoptosis. The MAM is now emerging as a site for ER to mitochondrial trafficking of cellular proteins and viral proteins. This project characterizes a cytomegalovirus (CMV) protein, known as UL37 exon 1 protein (pUL37x1) that targets the MAM, alters some of its functions, and traffics from the ER to mitochondria. This project will examine how proteins use the MAM to traffic from the ER to mitochondria. It is projected that CMV pUL37x1 trafficking uses MAM vesicular or tether mediated mechanisms to traffic to mitochondria. This project will test whether MAM tethers or vesicular components underlie ER to mitochondrial trafficking using complementary approaches of inhibition (siRNAs, knock out cells, dominant negative mutants, and chemical inhibitors) and overexpression of MAM tether or MAM vesicular pathway components. BROADER IMPACTSThese studies will impact the intracellular protein trafficking field, as well as education and society (technology development). The project aims to understand MAM organization and its regulation, which affects cellular physiology and responses. The use of multifocal structured illumination microscopy (MSIM) will advance the intracellular protein trafficking field and, more broadly, cellular imaging. The studies will include training of undergraduate researchers, and in particular will continue to support the efforts of the participating labs in training underrepresented minorities in science. Because of the multidisciplinary approaches employed in this project, students will benefit from training in confocal live cell imaging, MSIM imaging, and molecular virology and biochemical approaches, participating in scientific discussions, and presenting their work at scientific meetings.