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Developing yeast single cell microinjection technology for the study of protein

开发用于蛋白质研究的酵母单细胞显微注射技术

基本信息

批准号：
1666622
负责人：
金额：
--
依托单位：
University of Kent
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1666622
关键词：
Developing yeast single cell microinjection

项目摘要

The ability to maintain protein quality throughout the lifetime of a cell is crucial to the maintenance of longevity and healthy ageing. Loss of the ability to correctly manage protein quality control systems leads to the production of protein aggregates which can prove to be toxic and which are often linked to disease. The production of protein aggregates is complex and can result from mutation, a variety of cell stresses such as oxidative and heat stress, mitochondrial dysfunction, loss of chaperone activity or defects in process that can clear mis-folded protein products such as autophagy. To unravel the complexities that underlie protein quality control and its downstream effects many researchers turn to genetically tractable model organisms such as the budding yeast S. cerevisiae. Within this studentship we will conduct research that will facilitate the development of new technologies designed to improve and accelerate research into protein aggregation via collaboration with Singer Instruments. The project design offers a wide range of training within academic and industrial settings under an umbrella project that fits within the BBSRC strategic area of "Lifelong Health and Wellbeing".Project Overview:The student will focus upon the disease related aggregate forming protein superoxide dismutase 1 (SOD1). Mutations in SOD1 lead to familial Amyotrophic Lateral Sclerosis (ALS, Motor Neuron Disease) for which there is no known cure and for which the cause of cell death is currently unknown. The Sod1 protein is evolutionary conserved and mutant forms that are associated with ALS can be expressed in yeast leading to toxicity (our unpublished data). We are therefore able to utilise yeast as convenient platform to determine the nature of mutant Sod1 toxicity. The student will produce and purify mutant forms of Sod1 that are most commonly found in ALS patients using yeast expression systems and standard biochemical techniques. Purified Sod1 will then be induced to aggregate in vitro and microinjected into yeast cells using new technology being developed at Singer Instruments. This approach offers the unique advantage of being able to control the size of aggregates and concentration delivered to the cell at specific stages of the cell cycle. Following injection the health of cells will be monitored using a variety of assays that are well developed within our lab such as respiratory rate, ROS levels, viability and redox potential within specific compartments. Expression of mutant Sod1 and its toxicity will also be achieved by standard gene expression techniques. We will then micro-inject Sod1 aggregates into yeast cells lacking genes that control specific functions known to be involved in protein homeostasis and organelle function. This medium throughput will allow us to obtain new insights into the mode of Sod1 aggregate mediated toxicity. It will also provide a means to test exciting new microinjection technology that has tremendous potential and which will have many applications.Training:The students will receive the benefits of training opportunities in both the academic and industrial applicant labs. Within the academic environment the student will receive training in molecular biology techniques, live cell imaging, biochemical assays, protein purification and flow cytometry amongst others. At Singer Instruments the student will develop a different set of key skills that centre upon customised robotics, analytical software development and IT, quality control procedures and industrial standard lab protocols. The package represents an exciting opportunity that underpins cutting edge technology development and scientific research.

在细胞的整个生命周期中保持蛋白质质量的能力对于维持长寿和健康衰老至关重要。失去正确管理蛋白质质量控制系统的能力会导致蛋白质聚集体的产生，这些聚集体可能被证明是有毒的，并且通常与疾病有关。蛋白质聚集体的产生是复杂的，并且可以由突变、多种细胞应激（例如氧化应激和热应激）、线粒体功能障碍、伴侣蛋白活性的丧失或可以清除错误折叠的蛋白质产物（例如自噬）的过程中的缺陷引起。为了解开蛋白质质量控制及其下游效应的复杂性，许多研究人员转向遗传上易于处理的模式生物，如芽殖酵母S。酿酒的。在这个学生奖学金，我们将进行研究，这将促进新技术的开发，旨在改善和加速研究蛋白质聚集通过与歌手仪器合作。该项目设计提供了一个伞式项目下的学术和工业环境中的广泛的培训，适合在BBSRC的战略领域“终身健康和福祉”。项目概述：学生将专注于疾病相关的聚集体形成蛋白超氧化物歧化酶1（SOD 1）。SOD1的突变导致家族性肌萎缩性侧索硬化症（ALS，运动神经元疾病），目前还没有已知的治愈方法，细胞死亡的原因也是未知的。Sod1蛋白是进化保守的，与ALS相关的突变形式可以在酵母中表达，导致毒性（我们未发表的数据）。因此，我们能够利用酵母作为方便的平台来确定突变体Sod1毒性的性质。学生将使用酵母表达系统和标准生物化学技术生产和纯化ALS患者中最常见的Sod1突变形式。然后，纯化的Sod 1将在体外诱导聚集，并使用Singer Instruments正在开发的新技术显微注射到酵母细胞中。这种方法提供了能够控制在细胞周期的特定阶段递送至细胞的聚集体的大小和浓度的独特优势。注射后，将使用我们实验室内开发的各种检测方法监测细胞的健康状况，例如呼吸率，ROS水平，特定隔室内的活力和氧化还原电位。突变体Sod 1的表达及其毒性也将通过标准基因表达技术实现。然后，我们将微注射Sod 1聚合物到酵母细胞中，缺乏控制已知参与蛋白质稳态和细胞器功能的特定功能的基因。这种中等的通量将使我们能够获得新的见解的模式Sod 1聚集体介导的毒性。它还将提供一种手段来测试令人兴奋的新的显微注射技术，具有巨大的潜力，并将有许多应用。培训：学生将获得在学术和工业应用实验室的培训机会的好处。在学术环境中，学生将接受分子生物学技术，活细胞成像，生化分析，蛋白质纯化和流式细胞术等方面的培训。在Singer Instruments，学生将培养一套不同的关键技能，这些技能以定制机器人技术，分析软件开发和IT，质量控制程序和工业标准实验室协议为中心。该软件包代表了一个令人兴奋的机会，支持尖端技术开发和科学研究。