| Transfection and Activation of CofActor, a Light and Stress Gated Optogenetic Tool, in Primary Hippocampal Neuron Cultures
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Author:
Date:
2021-04-20
[Abstract] Proteins involved in neurodegeneration can be coupled with optogenetic reagents to create rapid and sensitive reporters to provide insight into the biochemical processes that mediate the progression of neurodegenerative disorders, including Alzheimer’s Disease (AD). We have recently developed a novel optically-responsive tool (the ‘CofActor’ system) that couples cofilin and actin (key players in early stage cytoskeletal abnormalities associated with neurodegenerative disorders) with light-gated optogenetic proteins to provide spatial and temporal resolution of oxidative and energetic stress-dependent biochemical events. In contrast to currently available small-molecule based biosensors for monitoring changes in the redox environment of the cell, CofActor is a ...
[摘要] [摘要]参与神经变性蛋白质可具有耦合光遗传学试剂来创建快速且灵敏的记者到provid Ë洞察介导的神经变性疾病,包括进展的生化过程阿尔茨海默氏病(AD)。我们最近开发了一种新型光学-响应工具(“辅”系统)夫妇COF伊林和行动中使用(与神经退行性疾病相关的早期阶段,细胞骨架异常关键球员)光门控光遗传学 蛋白质提供时空分辨率的氧化和高能应激依赖的生化事件。与目前可用的基于小分子的生物传感器来监测细胞氧化还原环境的变化相比,CofActor是一种光激活的,遗传编码的氧化还原传感器,可以通过精确的空间和时间控制来激活。在这里,我们描述了从新生小鼠制备的解离海马神经元培养物中CofActor系统的表达和激活的协议。将培养物转染用大号ipofectamine上的第五天体外(DIV5),然后暴露于细胞应激诱导刺激,导致的肌动蛋白的形成丝切蛋白可使用活细胞成像技术可以观察到杆。本文所述的方案可用于研究暴露于神经退行性刺激(例如毒性Aβ42低聚物)的活神经元中与压力相关的细胞骨架失调。此外,从AD的转基因小鼠模型和/或与KO相关的小鼠KO小鼠分离的神经元中传感器的表达可以促进我们对与神经变性相关的早期细胞骨架功能障碍的分子基础的理解。
[背景]神经变性疾病的生化标志(神经原纤维,团块和缠结,提高活性氧物质(ROS) ...
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| Mechanical Characterization of Glandular Acini Using a Micro-indentation Instrument
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Author:
Date:
2020-12-05
[Abstract] The linker of nucleoskeleton and cytoskeleton (LINC) complex is responsible for tethering the nucleus to the cytoskeleton, providing a pathway for the cell’s nucleus to sense mechanical signals from the environment. Recently, we explored the role of the LINC complex in the development of glandular epithelial acini, such as those found in kidneys, breasts, and other organs. Acini developed with disrupted LINC complexes exhibited a loss of structural integrity, including filling of the lumen structures. As part of our investigation, we performed a mechanical indentation assay of LINC disrupted and undisrupted MDCK II cells using a micro-indentation instrument mounted above a laser-scanning confocal microscope. Through a combination of force measurements acquired from the ...
[摘要] [摘要]的接头nucleoskeleton和细胞骨架(LINC)络合物负责核束缚到细胞骨架,提供用于细胞核从环境感测机械信号的通路。最近,我们探索了LINC复合物在腺上皮腺泡(例如在肾脏,乳房和其他器官中发现的腺泡)的发育中的作用。用破坏的LINC复合物开发的Acini表现出结构完整性的丧失,包括管腔结构的填充。作为研究的一部分,我们使用安装在激光扫描共聚焦显微镜上方的微型压痕仪对LINC破坏的和未破坏的MDCK II细胞进行了机械压痕测定。通过从微压痕仪获得的力测量值和从荧光图像获得的接触面积测量值的组合,我们确定了痤疮结构破裂的平均接触压力。在这里,我们提供了微压痕仪设计的详细描述,以及为执行这些生物压痕测量而开发的实验步骤。此外,我们讨论了确定痤疮结构破裂压力所需的数据分析步骤。尽管此协议专注于单个腺腺突的压痕,但此处介绍的方法可适用于针对2D和3D生物学系统执行各种机械压痕实验。
关键字:生物压痕,微压痕,生物力学,组织力学,核力学,LINC复合体,腺泡发育
[背景]生物压痕测试已成为手段长度尺度从亚细胞生物聚合物以多细胞组织结构来测量生物系统的材料性质。具有微米级探针和接触面积为10-100 nm 2的纳米压痕仪已用于测量生物聚合物和单个细胞的材料特性(Stolz等,2004; Sen等,2005; ...
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