| A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
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Author:
Date:
2020-12-20
[Abstract] Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes ...
[摘要] [摘要]圣RESS颗粒剂（SGS）是膜较少细胞器形式在通过相分离细胞质中，响应于不同的压力源。SG包含翻译停滞的mRNA，翻译中涉及的蛋白和各种RNA结合蛋白（RBP）。由于易于凝集的RBP的局部浓度很高，SG可能充当RBP异常相变的缩合位点，并且可能有助于形成在许多神经退行性疾病中发现的病理性细胞质内含物的固体蛋白质聚集体。大多数分析瞄准荷兰国际集团在研究限制性商业惯例的招募SG的基础上表达和SG招聘完整细胞的限制性商业惯例。但是，这些方法通常受到许多RBP的主要核定位的限制，这排除了足以进行SG定位的细胞质RBP浓度，并且没有解决独立于SG形成的RBP募集问题。在这里，我们提出了一个定量的方法来评估重组限制性商业惯例的募集至预先形成的SG，独立于RBP的核定位，用半透细胞和荧光显微镜。在该测定中，SG被第一LY由应激诱导的，然后第质膜ë应激的细胞随后被选择性地透化，以提供重组蛋白的访问的SG。通过用小麦胚芽凝集素阻塞核孔来阻止目的蛋白的核输入。这种测定方法使人们能够定量研究在没有核导入且在受控条件下将RBP募集到SG中的分子机制。该方法允许对野生型，突变体或直接比较翻译后修饰的限制性商业惯例，用于解决第其它蛋白质电子影响“预防或促进限制性商业惯例的SG关联，并且也适用于合成的肽。
图形摘要：
工作流概述在半SG招募重组蛋白质或肽的分析透化细胞 ...
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| Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
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Author:
Date:
2018-07-05
[Abstract] Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures.
[摘要] 培养细胞的免疫细胞化学是用于确定细胞结构内蛋白质的组成和定位的常用且有效的技术。 然而，传统的培养细胞固定和染色方案不能有效地保存培养的细胞色素，长期专门用于形态发生转运的丝状伪足。 结果，进行了有限的机械审讯以评估其监管。 我们开发了一种用于培养细胞的固定方案，该方案保留了细胞质，允许对内源性和过表达的蛋白质进行免疫荧光分析，这些蛋白质定位于脆弱的细胞结构。
【背景】Cytonemes被分类为薄的（~200nm直径）基于肌动蛋白的丝状伪足，长度超过2μm，可以转运形态发生素（Ramírez-Weber和Kornberg，1999）。这些信号结构首先在发育中的 Drosophila 翼成像盘中进行了详细分类和描述，随后在小鼠，小鸡和斑马鱼模型生物中进行了观察（Ramírez-Weber和Kornberg，1999; Sanders et al。，2013; Stanganello et al。，2015）。在大多数情况下，只有对过表达的荧光标记蛋白进行实时成像才能进行细胞色素检测。由于传统的固定方案未能保存这些脆弱的细丝，因此对培养细胞的细胞色素的检查受到限制。这些并发症一直是决定在发育和组织稳态期间驱动细胞色素形成和功能的细胞机制以及确定这些过程是否在疾病中被破坏的限制因素。
为了克服这些限制，我们开发了一种基于修饰电子显微镜固定剂（MEM-fix）的方案，该方案可以保留培养细胞的细胞质。 ...
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| Experimental Liver Fibrosis and Intrasplenic Transplantation of CD45+ Bone Marrow Cells
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Author:
Date:
2016-10-20
[Abstract] Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. Carbon tetrachloride-induced fibrosis (CCl4) and cirrhosis is one of the oldest, simplest and probably the most widely used toxin-based experimental model for the induction of fibrosis. Here we have explained experimental animal model of liver fibrosis using CCl4, injecting twice a week for a period of 8 weeks. In these fibrotic mice, bone marrow (BM) derived CD45+ cells were transplanted via intrasplenic route ...
[摘要] Liver fibrosis results from the excessive collagen deposition (collagen scar) by activated hepatic stellate cells (HpSCs), leading to the inhibition of normal liver regeneration and function. Fibrogenesis is a complex mechanism involving both the synthesis and degradation of matrix proteins by different cell types, mainly macrophages in the liver. Carbon tetrachloride-induced fibrosis (CCl4) and cirrhosis is one of the oldest, simplest and probably the most widely used toxin-based experimental model for the induction of fibrosis. Here we have explained experimental animal model of ...
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