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称为细胞器的内部隔室组成，它们具有不同的组成和功能。大多数细胞器含有称为脂质的脂肪样分子，它们形成限制膜，将细胞器内容物与细胞的其余部分以及许多类型的蛋白质分开。细胞器的功能需要输送新物质以及与细胞内其他细胞器进行物质交换。细胞器之间蛋白质和脂质的运输是由称为囊泡的小型球形载体介导的，囊泡从一个隔室中萌芽，并与其目的地隔室结合并融合以输送其内容物。这一过程在所有植物和动物中都是保守的，对于生命至关重要，如果出现缺陷，可能会导致人类出现大量疾病。病原细菌和病毒在其生命周期中也会利用它。囊泡运输具有高度特异性，因此囊泡在目的地隔间以选择性方式被识别，这确保它们将其内容物递送到正确的位置。尽管囊泡运输已经研究了几十年，但我们仍然对囊泡识别如何发生缺乏充分的了解。高尔基体是细胞中的主要运输枢纽。它从其他细胞器接收囊泡，并在囊泡中自己的子区室之间移动货物。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。高尔基体的囊泡识别是由称为高尔基蛋白的长蛋白介导的，它们的作用就像触手一样，在其末端捕获或束缚囊泡。先前的研究表明，高尔金以选择性的方式束缚囊泡，从而有助于高尔基体囊泡运输的特异性。然而，它们在囊泡上识别的内容尚不清楚。也不知道高尔金斯在囊泡识别中是否具有重叠的特异性。这项研究将解决这些悬而未决的问题，重点关注介导高尔基体内运输的高尔金斯，这对于该细胞器的功能至关重要。我们的初步数据表明，囊泡表面的脂质通过高尔金的选择性识别决定了高尔基体囊泡运输的特异性。为了检验这一假设，我们将使用纯化的脂质囊泡和高尔金斯，结合无偏脂质鉴定技术，研究高尔金斯的脂质结合特异性，并确定与脂质结合的高尔金斯的特征。随后，我们将确定高尔金-囊泡脂质相互作用在高尔基体货物蛋白运输和修饰中的重要性。这将通过基因编辑技术改变高尔金斯来实现，使它们不再结合囊泡脂质，并使用已建立的测定法评估对货物运输的影响。还将使用已建立的方法来评估货物修饰，以测量添加到高尔基体货物蛋白中的糖的数量和成分，这高度依赖于该细胞器的囊泡运输速率。为了在更生理的背景下评估高尔金与囊泡脂质结合的重要性，我们将在线虫秀丽隐杆线虫中进行类似的实验。将评估对发育、活力、组织形成和功能以及衰老的影响，同时分析不同细胞类型的高尔基体。由于该模型的遗传易处理性，我们还能够以不同的组合敲除或修饰高尔金斯，以评估这些蛋白质之间重叠特异性和功能冗余的程度，从而评估高尔基体囊泡运输的程度。这项工作对于我们理解囊泡运输，特别是如何实现囊泡运输的特异性非常重要，这是该领域一个尚未解答的主要问题。