Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers
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Author:
Date:
2017-02-05
[Abstract] Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.
[摘要] 属于类 Mollicutes 的数十种支原体物种作为细胞器在极点形成突起。它们通过细胞器与固体表面结合,并通过独特的机制沿着方向滑动,包括在宿主动物细胞上重复的结合,拉伸和释放与唾液酸化寡糖的循环。机械特征是了解传染病过程中涉及到的独特机制的关键信息。在本协议中,我们描述了一种测量由支原体(Mycoplasma)中最快的滑翔物种支原体移动产生的力的方法。该方案对于许多种滑动微生物的研究应该是有用的。
背景 表面运动系统分布在许多细菌物种上,与细菌鞭毛和真核细胞运动蛋白相比并不能很好地阐明(Jarrell和McBride,2008),尽管它们可能给我们提供了解细菌生存策略的关键信息。为了阐明动力机制,我们需要关于机械结构,能量流动以及包括速度和力在内的机械特性的信息。光学镊子是用于在显微镜下在微米范围内的微观操作或力测量的特殊方法,通过该技术,具有与介质不同的衍射指数的物体被捕获在聚焦激光束的中心(Ashkin等人,1986)。这种方法大大有助于阐明肌球蛋白,动力蛋白和驱动蛋白的运动系统的特征,现在已经成为生物物理学领域的一个方法。在这里,我们根据我们的研究(Miyata等人,2002; Tanaka等人,2016),提供了如何测量表面移动微生物产生的力的方案,对于M的滑翔机制。移动类 Mollicutes ...
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Lentiviral shRNA Screen to Identify Epithelial Integrity Regulating Genes in MCF10A 3D Culture
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Author:
Date:
2016-12-05
[Abstract] MCF10A 3D culture system provides a reductionist model of glandular mammary epithelium which is widely used to study development of glandular architecture, the role of cell polarity and epithelial integrity in control of epithelial cell functions, and mechanisms of breast cancer. Here we describe how to use shRNA screening approach to identify critical cell pathways that couple epithelial structure to individual cell based responses such as cell cycle exit and apoptosis. These studies will help to interrogate genetic changes critical for early breast tumorigenesis. The protocol describes a library of lentiviral shRNA constructs designed to target epithelial integrity and a highly efficient method for lentiviral transduction of suspension MCF10A cultures. Furthermore, protocols are ...
[摘要] MCF10A 3D文化系统提供了腺体乳腺上皮的还原剂模型,其广泛用于研究腺体结构的发育,细胞极性和上皮完整性在上皮细胞功能的控制中的作用以及乳腺癌的机制。在这里我们描述如何使用shRNA筛选方法来识别关键细胞通路,夫妇上皮结构到个别细胞的反应,如细胞周期退出和凋亡。这些研究将有助于询问对早期乳腺肿瘤发生至关重要的遗传变化。该协议描述了设计用于靶向上皮完整性的慢病毒shRNA构建体的文库和用于悬浮MCF10A培养物的慢病毒转导的高效方法。此外,提供的协议设置MCF10A 3D文化在Matrigel的形态和细胞反应研究通过结构化照明和共聚焦显微镜分析免疫染色的三维结构。 关键字: 3D文化,MCF10A,shRNA,上皮完整性,免疫荧光染色,3D成像,形态测量分析 [背景] 上皮细胞形成高度组织的组织结构,其提供物理支持和用于协调细胞信号传导的结构化支架。跨上皮结构的这种协调的信号传导对于上皮生物学是基本的;使得上皮细胞在调节器官大小,形状,功能和基于个体细胞的应答中的动态联合作用(Roignot等人,2013; Shamir和Ewald,2014)。上皮信号传导的联合指挥还提供了一种强有力的肿瘤抑制机制,通过将外部和内部有丝分裂信号门控到静止的上皮组织(Partanen等人,2013; Rejon等人 ...
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Isolation of Intestinal Mesenchymal Cells from Adult Mice
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Author:
Date:
2016-09-20
[Abstract] During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells ...
[摘要] 在过去20年间,肠间质细胞(IMC)已经作为重要的细胞类型出现,通过提供结构支持和生长调节元件在肠发育和体内平衡中起着中心作用。 IMC还积极参与伤口愈合反应,从而调节病理状况,例如组织修复,炎症,纤维化和癌发生(Powell等人,2011)。 我们最近已经证明肠间充质特异性信号在肠炎症和癌发生中在体内起重要的生理作用(Koliaraki等人,2012; Roulis等人,/em>。,2014; Koliaraki 。。,2015)。 在这里我们描述了用于从成年小鼠肠道分离和培养间充质细胞的酶法。
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