Determining the specificity of vesicle traffic at the Golgi apparatus

确定高尔基体囊泡运输的特异性

• 批准号: BB/X006859/1
• 负责人: Martin Lowe
• 金额: $ 69.27万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX006859%2F1
• 关键词: Determining; specificity; vesicle; traffic; Golgi

The cells that make up our organs and tissues are comprised of internal compartments, called organelles, that have distinct compositions and functions. Most organelles contain fat-like molecules called lipids that make a limiting membrane to separate the organelle contents from the rest of the cell, as well as many types of proteins. The function of organelles requires the delivery of new materials and the exchange of materials with other organelles in the cell. Transport of proteins and lipids between organelles is mediated by small spherical carriers called vesicles, which bud off one compartment and bind to and fuse with their destination compartment to deliver their contents. This process, which is conserved in all plants and animals, is essential for life, and when defective can result in a large number of diseases in humans. It is also exploited by pathogenic bacteria and viruses during their life cycle. Vesicle transport is highly specific, such that vesicles are recognized at the destination compartment in a selective manner, which ensures they deliver their contents to the correct place. Although vesicle transport has been studied for decades, we still lack a good understanding of how vesicle recognition occurs.The Golgi apparatus is a major transport hub in the cell. It receives vesicles from other organelles, and also moves cargo between its own sub-compartments in vesicles. Its major function is to sort cargo for distribution, and to modify it so it matures correctly. Vesicle recognition at the Golgi is mediated by long proteins called golgins, which are act like tentacles to capture, or tether, vesicles at their ends. Previous work has shown that golgins act in a selective manner to tether vesicles, thereby contributing to the specificity of vesicle transport at the Golgi. However, what they recognise on vesicles is not known. It is also not known whether the golgins have overlapping specificity in vesicle recognition. This study will address these outstanding questions, focussing on the golgins that mediate transport within the Golgi, which is critical for the function of this organelle. Our preliminary data suggests that lipids on the vesicle surface dictate the specificity of vesicle transport at the Golgi through selective recognition by the golgins. To test this hypothesis, we will investigate the lipid binding specificity of the golgins, using purified lipid vesicles and golgins, combined with unbiased lipid identification techniques, and determine the features of the golgins that bind to lipids. Subsequently, we will determine the importance of golgin-vesicle lipid interaction in the transport and modification of cargo proteins at the Golgi apparatus. This will be achieved using gene editing techniques to alter the golgins so they can no longer bind vesicle lipids, and effects upon cargo transport assessed using established assays. Cargo modification will be also assessed using established methods to measure the amount and composition of sugars added to the cargo proteins in the Golgi, which is highly dependent on vesicle transport rates at this organelle. To assess the importance of golgin binding to vesicle lipids in a more physiological context, we will perform similar experiments in the nematode worm C. elegans. Effects upon development, viability, tissue formation and function, and ageing, will be assessed alongside analysis of the Golgi in different cell types. Because of the genetic tractability of this model, we will also be able to knock-out or modify the golgins in different combinations to assess the extent of overlapping specificity and functional redundancy between these proteins, and hence of vesicle transport at the Golgi. The work will be important for our understanding of vesicle transport, and specifically in how the specificity of vesicle transport is achieved, which represents a major unanswered question in the field.

构成我们器官和组织的细胞由内部隔室组成，称为细胞器，具有不同的组成和功能。大多数细胞器含有脂肪样分子，称为脂质，它们构成限制膜，将细胞器内容物与细胞的其他部分以及许多类型的蛋白质分开。细胞器的功能需要新物质的传递以及与细胞内其他细胞器的物质交换。蛋白质和脂质在细胞器之间的转运是由称为囊泡的小球形载体介导的，囊泡从一个隔室中出芽，并与其目的隔室结合并融合以递送其内容物。这一过程在所有植物和动物中都是保守的，是生命所必需的，如果有缺陷，可能导致人类大量疾病。它也被致病细菌和病毒在其生命周期中利用。囊泡运输是高度特异性的，使得囊泡以选择性的方式在目的地隔室被识别，这确保它们将其内容物递送到正确的位置。尽管人们对囊泡运输的研究已经有几十年的历史，但是对于囊泡识别的过程仍然缺乏深入的了解。它从其他细胞器接收囊泡，并在囊泡中的子隔室之间移动货物。它的主要功能是对货物进行分类以进行分配，并对其进行修改以使其正确成熟。囊泡在高尔基体的识别是由一种叫做golgins的长蛋白介导的，这种长蛋白就像触角一样捕获或拴系囊泡的末端。以前的工作表明，高尔基体以选择性的方式拴系囊泡，从而有助于囊泡在高尔基体运输的特异性。然而，它们在囊泡上识别什么尚不清楚。也不知道golgins是否在囊泡识别中具有重叠特异性。这项研究将解决这些悬而未决的问题，集中在高尔基体内，这是这个细胞器的功能至关重要的介导运输的golgins。我们的初步数据表明，囊泡表面的脂质决定了囊泡运输的特异性，通过选择性识别的高尔基体。为了验证这一假设，我们将调查的golgins的脂质结合特异性，使用纯化的脂质囊泡和golgins，结合无偏的脂质鉴定技术，并确定的golgins结合脂质的功能。随后，我们将确定在高尔基体的货物蛋白的运输和修改的重要性，高尔基体囊泡脂质相互作用。这将使用基因编辑技术来改变golgins，使它们不再结合囊泡脂质，并使用已建立的测定评估对货物运输的影响。货物修改也将使用已建立的方法来评估，以测量添加到高尔基体中的货物蛋白的糖的量和组成，这是高度依赖于囊泡运输率在这个细胞器。为了评估高尔基体结合囊泡脂质的重要性，我们将在线虫C。优美的对发育、活力、组织形成和功能以及衰老的影响将与不同细胞类型中高尔基体的分析一起进行评估。由于该模型的遗传易处理性，我们也将能够敲除或修饰不同组合的高尔基体，以评估这些蛋白质之间的重叠特异性和功能冗余的程度，从而评估高尔基体的囊泡运输。这项工作对于我们理解囊泡运输，特别是囊泡运输的特异性是如何实现的，这是该领域一个尚未回答的主要问题，将是重要的。