High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
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Author:
Date:
2018-01-05
[Abstract] Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017).
[摘要] 在这里我们描述了两种测定方法来测量神经内分泌细胞中的致密核心囊泡(DCV)胞吐作用介导的货物分泌。 为了在受调控的DCV胞吐作用中对新基因进行siRNA筛选,我们开发了使用DCV货物,NPY-金星以及正交3H-血清素分泌测定的基于读板器的分泌测定法。 NPY-金星分泌测定成功地用于高通量siRNA筛选,并且使用血清素分泌测定来验证从筛选中鉴定的命中(Sorensen,2017; Zhang等人,2017)。
【背景】致密核心囊泡(DCV)胞吐作用介导内分泌和神经内分泌细胞分泌蛋白质,肽和小分子。当蛋白质和肽激素被内质网上的核糖体合成时,蛋白质和肽激素进入分泌途径,并在跨高尔基网络(TGN)(Borgonovo等人,2006; Bowman等, >等人,2009)。小分子物质,如5-羟色胺,直接被胞质单胺转运蛋白(VMATs)从细胞质直接吸收到DCV的内腔(Sudhof,2004)。在非刺激条件下,货物分子被储存在靠近质膜的DCV内。当细胞接受增加细胞质Ca ...
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Isolation of Cytosol, Microsome, Free Polysomes (FPs) and Membrane-bound Polysomes (MBPs) from Arabidopsis Seedlings
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Author:
Date:
2017-08-05
[Abstract] The plant endomembrane system plays vital roles for synthesis, modification and secretion of proteins and lipids. From the classic view, only mRNAs encoding secreted proteins could be targeted to the endoplasmic reticulum (ER) for translation via a co-translational translocation manner, however, recently this model has been challenged by accumulative evidence that lots of cytosolic mRNAs could also associate with ER, and that some categories of small RNAs are enriched on ER. These results suggested unrevealed functions of ER beyond our current knowledge. The large scale identification of RNAs and proteins on microsome is crucial to demonstrating the ER function and the studies will be boosted by next generation sequencing technology. This protocol provides a technical workflow to isolate ...
[摘要] 植物内膜系统对蛋白质和脂质的合成,修饰和分泌起着至关重要的作用。 从经典观点来看,只有编码分泌蛋白质的mRNA才能通过协同翻译方式靶向内质网(ER)进行翻译,然而最近,这一模型已经被大量的细胞溶质mRNA也可能与 ER,并且一些类别的小RNA在ER上富集。 这些结果表明ER的功能超出了目前的知识。 在微粒体上大规模鉴定RNA和蛋白质对于显示ER功能至关重要,研究将由下一代测序技术提升。 该协议提供了从植物组织中分离细胞质,微粒体,游离多聚体(FP)和膜结合多聚体(MBP)的技术工作流程。 分离的级分适用于mRNA,小RNA和蛋白质的基因组广谱分析。 【背景】植物内膜系统对于细胞壁形成,脂质生物合成,蛋白质合成,修饰,折叠和贩运非常重要。根据共翻译易位模型,分泌蛋白N末端的信号肽由细胞溶质多核糖体合成,然后由ER上的信号识别粒子识别,其余蛋白质部分随后在ER上合成。根据该模型,只有编码分泌蛋白的mRNA可以被带到ER进行翻译(Peter和Johnson,1994)。然而,从哺乳动物和植物细胞ER(Lerner等人,2003; de ...
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Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies
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Author:
Date:
2017-04-20
[Abstract] Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.
[摘要] 军团菌具有关键的分泌机制,以将毒力因子递送至真核宿主细胞。在本协议中,我们描述了从L / L分离Dot / Icm IV型分泌系统的天然核心复合物的步骤。肺炎支原体旨在进行生物化学和透射电子显微镜分析。
嗜肺军团菌是革兰氏阴性细菌病原体,其导致被称为退伍军人病的肺部感染(Fields等人,2002)。 L。嗜肺杆菌利用由 dot / cm 基因编码的IV型分泌系统(T4SS)将大约300种细菌蛋白转运到其真核宿主细胞质中以劫持细胞过程(Hubber和Roy, 2010)。由超过20种蛋白组成,T4SS是建立在细菌内膜和外膜上的纳米机器(Nagai和Kubori 2011; Kubori和Nagai 2016)。 Dot / Icm T4SS的核心复合物是系统的生物化学稳定部分,并形成桥接内膜和外膜的输送导管(Kubori等人,2014)。核心复合物由至少五种蛋白质组成;三种外膜相关蛋白,DotC,DotD和DotH,以及两种内膜蛋白DotF和DotG(Vincent等人,2006)。基于来自鼠伤寒沙门氏菌的另一种细菌纳米机械的III型分泌系统的生物化学分离方法(Kubori等人,1998; Marlovits等人, ...
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