Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides,ACS Biomaterials Science & Engineering

当前位置： X-MOL 学术 › ACS Biomater. Sci. Eng. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides
ACS Biomaterials Science & Engineering ( IF 5.4 ) Pub Date : 2021-01-14 , DOI: 10.1021/acsbiomaterials.0c01117
David R Tyrpak ₁ , Yaocun Li ₁ , Siqi Lei ₁ , Hugo Avila ₁ , John Andrew MacKay _{1,

2,

3}

Affiliation

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (T_t). To do so, this Method paper describes a simple live-cell imaging technique to estimate the T_t of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different T_t, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower T_t. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular T_t of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

中文翻译：

细胞内弹性蛋白样多肽转变温度的单细胞定量

弹性蛋白样多肽 (ELP) 是模块化的刺激响应材料，可响应加热而自组装成富含蛋白质的微结构域。通过将 ELP 克隆到效应蛋白，表达的细胞内融合甚至可以调节细胞途径。设计这些融合体的关键步骤是确定和控制它们的细胞内相变温度 ( T _t )。为此，本文描述了一种简单的活细胞成像技术，通过与荧光 ELP 标记物共转染来估计非荧光 ELP 融合蛋白的T _t 。然后，通过非荧光和荧光 ELP 融合蛋白的共同组装，可以在活细胞中可视化细胞内微结构域的形成。如果两个ELP融合体具有不同的T _t ，则细胞内ELP混合物在与具有较低T _{t 的}融合相对应的温度下相分离。此外，与单一转染处理相比，共组装的 ELP 微结构域通常在大小或数量上表现出明显的差异。这些功能使活细胞成像实验和图像分析能够确定相关 ELP 融合库的细胞内T _t 。作为案例研究，我们采用最近报道的 Caveolin1-ELP 库 (CAV1-ELP)。除了提供详细的协议之外，我们还报告了一个名为 SIAL（简单图像分析库）的有用 FIJI 插件的开发，其中包含用于图像随机化和致盲、表型评分和 ROI 选择的程序。这些任务是此处详细介绍的协议的重要部分，也常用于其他图像分析工作流程。

更新日期：2021-02-08

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11