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.

组成我们的器官和组织的细胞由具有不同组成和功能的内部隔室（称为细胞器）组成。大多数细胞器都包含称为脂质的脂肪分子，这些分子是限制膜，可以将细胞器含量与细胞的其余部分以及许多类型的蛋白质分开。细胞器的功能需要提供新材料并与细胞中其他细胞器的材料交换。细胞器之间蛋白质和脂质的转运是由称为囊泡的小球形载体介导的，该载体从一个隔间中芽开出，并与其目的地隔间结合并融合并融合其含量。这个过程在所有动植物中都保守，对生命至关重要，并且有缺陷会导致人类大量疾病。在生命周期中，病原细菌和病毒还可以利用它。囊泡的传输是高度特异性的，因此以选择性的方式在目的地隔室中识别囊泡，以确保它们将其内容物传递到正确的位置。尽管已经研究了数十年的囊泡运输，但我们仍然缺乏对囊泡识别方式的很好的了解。高尔基体是该细胞中主要的运输中心。它从其他细胞器接收囊泡，还可以在囊泡中自身的子街区之间移动货物。它的主要功能是对货物进行分配，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Golgins的长蛋白介导的，这些蛋白质的作用像捕获或系绳的触手。先前的工作表明，戈尔金斯以选择性的方式对串线囊泡起作用，从而有助于高尔基体在囊泡运输的特异性。但是，他们在囊泡上认识到的尚不清楚。还不知道戈尔金人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杰出的问题，重点是介导高尔基体内传输的高尔金，这对于该细胞器的功能至关重要。我们的初步数据表明，囊泡表面上的脂质通过高尔金的选择性识别来决定高尔基体在高尔基体的特异性。为了检验这一假设，我们将使用纯化的脂质囊泡和戈尔金斯（Golgins）和戈尔金（Golgins），与无偏的脂质识别技术结合，并确定与脂质结合的高尔金（Golgins）的特征。随后，我们将确定高尔金脂质相互作用在高尔基体货物蛋白转运和修饰中的重要性。这将是使用基因编辑技术来改变Golgins的，因此它们无法再结合囊泡脂质，并使用既定测定法评估了对货物运输的影响。还将使用既定方法来评估货物修饰，以测量高尔基体中添加到货物蛋白中添加到货物蛋白的糖的量和组成，该糖蛋白高度依赖于该细胞器的囊泡传输速率。为了评估在更生理的环境下戈尔金与囊泡脂质结合的重要性，我们将在线虫蠕虫秀丽隐杆线虫中进行类似的实验。将在不同细胞类型的高尔基体分析的同时评估对发育，生存力，组织形成和功能以及衰老的影响。由于该模型的遗传障碍性，我们还将能够以不同组合的形式敲除或修改Golgins，以评估这些蛋白质之间的重叠特异性和功能冗余的程度，因此在Golgi上的囊泡转运。这项工作对于我们对囊泡运输的理解至关重要，特别是在如何实现囊泡运输的特异性方面，这代表了该领域的主要未解决问题。