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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Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
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Author:
Date:
2017-08-05
[Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of ...
[摘要] 集群定期间隔短回归重复(CRISPR)和CRISPR相关(Cas)系统作为细菌和古菌中的适应性免疫系统,用于防御入侵病毒和质粒(Barrangou和Marraffini,2014)。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向(Jinek et al。,2012; Cong等人,2013; Mali ,2013; Zetsche等人,2015)。然而,真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9(SpCas9)靶向DNA接近的障碍,并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ (Knight等人,,2015; Hinz等人,2015; Horlbeck等人,2016年) ; ...
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iPS Cell Induction from Human Non-T, B cells from Peripheral Blood
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Author:
Date:
2013-09-20
[Abstract] The generation of iPS cells gives an opportunity to use patient-specific somatic cells which are a valuable source for disease modeling and drug discovery. To promote these studies, it is important to make iPS cells from easily accessible and less invasive tissues like blood. Here, we describe the basic method to generate human iPS cells from adult peripheral blood. After the isolation of mononuclear cells, a combination of cytokines stimulates the expansion of hematopoietic stem/progenitor population, which is the main target of this protocol. The cells are transduced with plasmid mixture encoding reprogramming factors. In most cases, the plasmids are lost during the establishment of iPS clones.
[摘要] iPS细胞的产生提供了使用患者特异性体细胞的机会,其是用于疾病建模和药物发现的有价值的来源。 为了促进这些研究,重要的是使iPS细胞从容易获得和侵入性较小的组织如血液。 在这里,我们描述从成人外周血生成人类iPS细胞的基本方法。 在分离单核细胞后,细胞因子的组合刺激造血干/祖细胞群的扩增,这是该方案的主要目标。 用编码重编程因子的质粒混合物转导细胞。 在大多数情况下,质粒在iPS克隆的建立期间丢失。
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