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Developing qPAINT to count molecules in polarity complexes and measure secretory cargo flux in epithelial cells.

开发 qPAINT 以对极性复合物中的分子进行计数并测量上皮细胞中的分泌货物通量。

基本信息

批准号：
BB/V008595/1
负责人：
Daniel St Johnston
金额：
$ 65.74万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FV008595%2F1
关键词：
Developing qPAINT count molecules polarity

项目摘要

The aim of this project is to develop and validate a new microscopy technique called qPAINT that counts the number of protein molecules in specific structures or complexes inside a cell. This is important, because it can reveal where proteins work together to perform biological functions and will provide quantitative data on how many copies of each protein a structure contains, which is essential for understanding how these proteins interact and for building models of biological processes. To test qPAINT, we will count the number of copies of specific proteins in the nuclear pores, which are the channels that regulate the movement of molecules in and out of the cell nucleus. We have chosen the nuclear pore because it is one of the few structures in the cell where the number of copies of each protein is already known. This will allow us to verify that qPAINT produces accurate data and to calibrate our system so that we can count the number of molecules in other structures whose composition is unknown.Once validated, we will use qPAINT to analyse the spatial organisation of epithelial cells, the most common cell-type in our bodies. Epithelial cells stick together to form the sheets or tubes that make up most of our organs, where they act as barriers between compartments (e.g. blood vessels; secretory glands) or between the inside and outside of the body (skin, digestive system and lungs). The formation of epithelial sheets depends on the coordinated polarisation of the cells, so that all have the same surface (apical) facing the outside and the inside (basal). Epithelial cells also control the movement of molecules from one side of the sheet to the other. For example, intestinal cells absorb food from the gut, while the cells of the mammary gland secrete milk proteins apically into mammary ducts. Disruption of apical-basal polarity can lead to several types of disease. For example, more than 80% of cancers arise from epithelial tissues and one of their hallmarks is a progressive loss of polarity, which correlates with tumour malignancy. The polarity proteins that make the apical and basal sides of epithelial cells different are largely known, but we have limited information about where these proteins interact with each other and how much of each protein is present. Recent research also suggests that some of these polarity proteins form clusters, but the mechanisms that drive cluster formation are unclear. We will investigate how these clusters form using qPAINT to count the number of molecules of each polarity protein in clusters in different regions of the cell, as well as their overall levels in the cytoplasm. Another key feature of epithelial cells is their ability to target the secretion of proteins to different sides of the cell. Secreted proteins are made in the endoplasmic reticulum and then move to the Golgi complex, where they are packaged into small vesicles surrounded by lipid membranes that are transported to the cell surface. We have developed a system in which we can release a pulse of a labelled secreted protein from the endoplasmic reticulum and watch its movement to the cell surface. We now plan to use qPAINT to count the number of molecules in the Golgi, vesicles and at the cell surface at different times after release. This will allow us to calculate how many cargo molecules are packaged into a vesicle and how quickly each secreted cargo moves to the cell surface. By releasing two cargoes that traffic to different places simultaneously, we will also measure how and where secreted proteins are sorted into the vesicles that deliver them to the correct region of the cell surface. In conclusion, this project will advance our quantitative understanding of how cells are polarised and how the secretory system sorts and transports proteins to the cell surface, while demonstrating how qPAINT can be used to count the number of molecules in any structure or region of interest in a cell.

该项目的目的是开发和验证一种名为qPAINT的新显微镜技术，该技术可以计算细胞内特定结构或复合物中蛋白质分子的数量。这一点很重要，因为它可以揭示蛋白质在哪里共同发挥生物学功能，并提供关于每个蛋白质结构包含多少拷贝的定量数据，这对于理解这些蛋白质如何相互作用和构建生物过程模型至关重要。为了测试qPAINT，我们将计算核孔中特定蛋白质的拷贝数，核孔是调节分子进出细胞核的通道。我们之所以选择核孔，是因为它是细胞中已知每种蛋白质拷贝数的少数结构之一。这将使我们能够验证qPAINT产生准确的数据，并校准我们的系统，以便我们可以计算其他结构中组成未知的分子数量。一旦验证，我们将使用qPAINT分析上皮细胞的空间组织，这是我们体内最常见的细胞类型。上皮细胞粘在一起形成构成我们大部分器官的薄片或管，在那里它们充当隔室之间的屏障（例如血管;分泌腺）或身体内外（皮肤，消化系统和肺）之间的屏障。上皮片层的形成取决于细胞的协调极化，因此所有细胞都具有面向外部和内部（基底）的相同表面（顶端）。上皮细胞还控制分子从片材的一侧到另一侧的运动。例如，肠细胞从肠道吸收食物，而乳腺细胞将乳蛋白分泌到乳腺导管顶端。顶端-基底极性的破坏可导致几种类型的疾病。例如，超过80%的癌症来自上皮组织，并且它们的标志之一是极性的进行性丧失，这与肿瘤恶性程度相关。使上皮细胞的顶侧和基底侧不同的极性蛋白质在很大程度上是已知的，但我们对这些蛋白质在哪里相互作用以及每种蛋白质存在多少的信息有限。最近的研究还表明，这些极性蛋白中的一些形成簇，但驱动簇形成的机制尚不清楚。我们将使用qPAINT来研究这些簇是如何形成的，以计数细胞不同区域簇中每种极性蛋白的分子数量，以及它们在细胞质中的总体水平。上皮细胞的另一个关键特征是它们能够将蛋白质分泌到细胞的不同侧面。分泌的蛋白质在内质网中产生，然后移动到高尔基复合体，在那里它们被包装成被脂质膜包围的小泡，这些小泡被运输到细胞表面。我们已经开发了一个系统，在这个系统中，我们可以从内质网释放一个标记的分泌蛋白脉冲，并观察它向细胞表面的运动。我们现在计划使用qPAINT来计算释放后不同时间高尔基体、囊泡和细胞表面的分子数量。这将使我们能够计算有多少货物分子被包装到囊泡中，以及每种分泌的货物移动到细胞表面的速度。通过同时释放两种运输到不同地方的货物，我们还将测量分泌的蛋白质如何以及在哪里被分选到囊泡中，将它们运送到细胞表面的正确区域。总之，该项目将推进我们对细胞如何极化以及分泌系统如何将蛋白质分类和运输到细胞表面的定量理解，同时展示qPAINT如何用于计算细胞中任何结构或感兴趣区域的分子数量。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A single-molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila.

DOI：
10.1242/jcs.259570
发表时间：
2021-12-15
期刊：
Journal of cell science
影响因子：
4
作者：
Cheng J;Allgeyer ES;Richens JH;Dzafic E;Palandri A;Lewków B;Sirinakis G;St Johnston D
通讯作者：
St Johnston D

User-friendly Oblique Plane Microscopy on a fully functional commercially available microscope base

用户友好的斜平面显微镜，位于功能齐全的商用显微镜底座上