小鼠模型的验证扰乱LINC配合物在细胞特异性的方式
Summary
Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.
Abstract
Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.
Introduction
核信封（NE）分隔细胞质核质。它是由内和外核膜（INM和ONM，分别）连接在核孔。由两种膜划定的管腔被称为核周腔（PNS）。所述ONM是粗面内质网（ER）的延伸，并且INM粘附到核层，核型-V的中间体长丝的小梁由A-和B-型表示核纤层蛋白1,2-。的核骨架，骨架（LINC）配合的接头是跨越整个核膜物理细胞核的内部连接到细胞骨架细丝和分子马达（图1A）的大分子装配体。它们由进化上高度保守性表征的网元积分跨膜蛋白的两个家庭之间的相互作用的：太阳（Sad1在每个/ Unc84）的蛋白质和Nesprins（核膜SPectRINS）。在哺乳动物中，SUN1和SUN2芳在INM，其N端核质区域直接与A-和B型的核纤层蛋白3-5交互电子跨膜蛋白。在INM的另一边，PNS内，孙海港蛋白质进化保守舒展〜150℃末端氨基酸称为孙域。孙域直接交互与进化保守KASH（Klarsicht / ANC-1，地久天长同源）域，Nesprins的分子信号。 KASH域由一个伸展的30〜C末端氨基酸突入PNS后跟一个跨膜结构域6的。至少有四个不同的Nesprin基因（Nesprin1-4）编码KASH-包含定位于东北7蛋白质。 Nesprins，其大小各不相同的50kDa〜（Nesprin4）的一个惊人1000 kDa的（Nesprin1巨型）胞质区，包含多个血影重复以及特定的图案使它们的相互作用与细胞骨架组分如肌动蛋白，网蛋白和分子马达8- 13。
<P类=“jove_content”>研究脊椎动物和无脊椎动物表明，核纤层蛋白/阳光/ Nesprin /分子马达构成一个进化上保守的“轴”控制核迁移和锚地。几个敲除（KO）小鼠的LINC复杂的组件的模型已经描述并均有助于提供一个框架哺乳动物发育9,14,15期间理解Sun和Nesprin蛋白质在网元的角色。然而，这些模型本几个显著缺陷，最明显的是：1）难以在解释由于细胞中区分的表型贡献非自主的效果，2）表型困难KASH含与KASH少Nesprin异构体16，3）的Sun和Nesprin蛋白在多种细胞类型的网元功能冗余需要复杂的育种方案来灭活所有SUN-KASH相互作用小鼠17只小鼠中4）围产期杀伤力不足两个的KASH结构域Nesprins1和2排除成人的分析表型18。这个协议描述了一种新的小鼠模型旨在破坏体内所有SUN-KASH相互作用，在一个细胞中自主和发育调节的方式，从而绕过许多上面列出的缺点。此酶Cre /液氧系小鼠模型依赖于两个重要的概念：1）任何已知Nesprin蛋白的KASH域足以靶向EGFP到在细胞培养系统中的网元，2），SUN域相互作用混乱与KASH域，从而过度任何KASH结构域将饱和所有的内源性SUN结构域和灭活LINC配合的显性负方式17（ 图1B）。本协议描述的组织采伐和用于确认在小脑浦肯野细胞都SUN-KASH相互作用的中断处理步骤。
Protocol
Representative Results
Discussion
最关键的步骤，以成功地研究LINC配合物在体内使用的Tg（CAG-LacZ的/ EGFP-KASH2）模型是确定一个合适的Cre小鼠路线的作用。实际上，如果是的Cre活性在其他细胞类型参与类似的途径，它可以复杂化结果的解释。因此，为了检查附近小区是重要的，如在小脑（图2B）的分子和颗粒细胞层中所示此处。同样，为了最大限度地提高任何生理相关表型它以识别的Cre应变横跨感兴趣的细胞类?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢分子遗传学核心和小鼠遗传学核心的华盛顿大学医学院圣路易斯学院（系眼科及视觉科学学院）的形态和成像的核心工作人员。作者是由麦克唐纳中心细胞与分子神经生物学，希望中心神经疾病的小额赠款计划的支持下，国家眼科研究所（＃R01EY022632卫生署），国家眼科研究所中心核心格兰特（＃P30EY002687）和不受限制的赠款防盲研究眼科及视觉科学系。
Materials
| Sucrose | Sigma Aldrich | S0389 | |
| 10x PBS | Gibco | 14200-075 | |
| 16% Paraformaldehyde Solution | Electron Microscopy Sciences | 15710 | |
| OCT Compound | Tissue-Tek | 4583 | |
| Adhesion Slides | StatLab | M1000W | |
| Donkey Serum | Sigma Aldrich | D9663 | |
| Triton X-100 | Sigma Aldrich | T9284 | |
| ImmuEdge Pen | Vector Laboratories | H-4000 | |
| Anti-Calbindin Antibody | Sigma Aldrich | C9848 | |
| Anti-EGFP Antibody | Abcam | ab13970 | |
| Anti-Nesprin2 Antibody | Previously described in Ref. 21 | ||
| Fluorescent Mounting Media | Dako | S3023 | |
| 2-methyl butane | Sigma Aldrich | O3551 |
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