| Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells
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Author:
Date:
2021-02-05
[Abstract] Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased ...
[摘要] [摘要]连接蛋白是有助于直接和局部旁分泌通过自己的能力介导的细胞-细胞通信膜结合蛋白寡聚成六聚半通道。当相邻通道对齐时,它们形成间隙连接,为单元之间的信息传输提供直接路径。与通常在生理条件下打开的完整的间隙连接相反,未对接的半通道的打开概率较低,主要是在受伤时打开。半通道允许小分子和离子(约1kDa)释放到局部细胞间环境中,并且过度表达/活性 与许多疾病状况有关。羧基荧光素染料的摄取量度了半通道的功能性表达,其中增加的半通道活性/功能反映了增加的负荷。该技术依赖于通过开放性半通道对不可渗透膜的荧光示踪剂的吸收,可用于比较在不同条件下(例如对照与疾病)培养的细胞单层之间的通道活性。其他技术(例如生物素化和电生理学)可以分别测量细胞表面表达和半通道开放可能性,但是,羧基荧光素的摄取提供了一种简单,快速且经济高效的方法来测定多种细胞类型的体外半通道活性。
图形摘要:
使用染料吸收量度半通道活性
[背景]连接蛋白(CX )是一体的跨膜蛋白,其寡聚到连接子在细胞表面。连接子与相似的六聚体蛋白复合物停靠在相邻细胞上,形成用于间隙连接细胞间通讯的双向导管(GJIC;Bosco等人,2011 ...
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| Real-time Three-dimensional Tracking of Endocytic Vesicles
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Author:
Date:
2020-10-20
[Abstract] Endocytic trafficking and recycling are fundamental cellular processes that control essential functions such as signaling protein complexes transport and membrane identity. The small GTPase Rabs are indispensable component of the endosomal recycling machinery. The Rabs bind to effectors to mediate their functions, such as protein sorting and degradation, membrane tethering or lipid modification, and organelle motility. Due to the complex and dynamic nature of endosomal compartments and tracking route, detailed multiparametric analyses of three-dimensional data by quantitative methods are challenging. Here, we describe a detailed time-lapse imaging protocol designed for the quantitative tracking of single endosomal vesicles, using GFP-Rab4-positive recycling endosomes. This method permits ...
[摘要] [摘要]内吞运输和再循环是基本的细胞过程,它们控制诸如信号蛋白复合物运输和膜特性等基本功能。小GTPase-Rabs是内质体回收机械中不可缺少的组成部分。Rabs结合效应器介导其功能,如蛋白质的分类和降解,膜栓系或脂质修饰,以及细胞器的运动。由于内体隔室和追踪路线的复杂性和动态性,用定量方法对三维数据进行详细的多参数分析是一项具有挑战性的工作。在这里,我们描述了一个详细的延时成像协议,设计用于定量跟踪单个内囊泡,使用GFP-Rab4阳性循环内体。这种方法允许在三维活体细胞成像中自动跟踪单个内吞小泡,允许研究多个参数,如丰度、速度、方向性、亚细胞定位以及蛋白质共定位。该协议可广泛应用于各种环境下的细胞模型,包括生长因子刺激、基因敲除、药物治疗等,适用于高通量筛选。 [背景] 越来越多的证据强调了在细胞迁移、粘附、形态发生、增殖、胞质分裂以及学习和记忆等不同过程中协调的内质体再循环的重要性(Grant和Donaldson,2009年;Parachoniak和Park,2012年;Wandinger Ness和Zerial,2014年;Zaoui等人,2019a和2019b). 哺乳动物中有70多种Rab-gtpase,它们在膜转运中具有不同的定位和功能。更复杂的是,虽然大多数Rab-gtpase是普遍存在的,但有些表现出组织特异性表达(van der ...
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