Name: live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem
Uploaded: 2020-05-23T14:30:00
Description: Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

1. 植物生长
<ol><li>播种阿拉比多普斯种子表达微管结合域与绿色荧光蛋白（MBD-GFP）10在土壤上，并保持在漫长的一天（16小时白天/8小时晚上），20°C/6°C条件1周的发芽。</li>
	<li>发芽后，将幼苗转移到具有足够生长空间的新盆栽，使植物生长强劲。保持植物在短天（8小时白天/16小时晚上），20°C/16°C条件3-5周。</li>
	<li>将植物转移到漫长的一天（16小时白天/8小时夜间），...

<ol>
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机械信号转导事件的评估对于识别机械感知和转导通路所涉及的分子调节器至关重要。此处描述的协议通过使用皮层微图响应作为 Arabidopsis SAM 中此类过程的读出，提供了此类事件的定量视图。这里描述的程序经常用于各种组织类型16，17，18，19的多个研究。在所有组织类型中，微管各向异性显...

植物细胞被多糖和糖蛋白的细胞外基质包围，这些基质在机械上类似于一种纤维增强复合材料，能够动态地改变其机械性能1。植物细胞的生长是由水吸收到细胞中所推动的，这导致细胞壁上同时积聚的拉伸力。为了响应这种力，对细胞壁的物理状态的修改允许细胞膨胀。与含有细胞的二级细胞壁相比，具有原壁的细胞能够快速生长，这主要是由于其中多糖的化学成分不同。原发性壁细胞由纤维素、血脂素和果胶以及缺乏木质素组成，木质素存在于二级细胞壁2中。纤维素是一种通过β-1，4键连接的葡萄糖聚合物，是细胞壁的主要成分。它被组织成纤维结构，能够承受细胞生长3过程中经历的高拉伸力。除了承受拉伸力外，沿优先方向进行机械加固还会导致沿垂直于纤维素微纤维的净向轴的涡轮驱动膨胀。纤维素微纤维的组织受皮质微管细胞骨架的影响，因为它们引导位于血浆膜4的纤维素合成复合物的方向运动。因此，使用微管相关蛋白质或与荧光分子融合的块状蛋白监测皮质微管组织，可作为观察植物细胞中纤维素上表面模式的代理。
皮质微管细胞骨架的图案在细胞和组织形态衍生的机械力的控制之下。皮质微管组织在位于SAM顶点的细胞中没有任何优先组织，而处于外围的细胞和SAM与新兴器官之间的边界则有一个稳定、高度组织的皮质微管5的超细胞阵列。已经开发出几种方法来物理干扰细胞的机械状态。渗透状态的变化，以及对影响细胞壁刚度的药理和酶化合物的治疗，都可能导致细胞6，7经历的拉伸力的后续变化。使用允许组织经历的压缩力逐渐增加的装置是另一种选择。离心力的应用也被证明对机械力有影响，而与细胞9没有任何物理接触。然而，在一组细胞中改变定向力的最广泛使用的方法利用了以下事实：所有表皮细胞都在紧张状态，细胞的物理消融将消除局部的结节压力，并破坏细胞对细胞的粘附，从而改变相邻细胞经历的拉伸力。这通过瞄准高功率脉冲紫外激光或通过细针执行。
在这里，我们详细阐述了在SAM的机械扰动成像和评估皮质微管行为的过程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FibrilTool</td>
			<td></td>
			<td></td>
			<td>Boudaoud, A. et al., Nat Protoc. 2014</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td></td>
			<td>Schindelin, J. et al., Nat Methods. 2012</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Merck</td>
			<td>1.04201.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td>DM6000 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP4-GFP</td>
			<td></td>
			<td></td>
			<td>Marc, J. et al., Plant Cell 1998</td>
		</tr>
		<tr>
			<td>micropore tape</td>
			<td>Leukopor</td>
			<td>02482-00</td>
			<td></td>
		</tr>
		<tr>
			<td>MorphographX</td>
			<td></td>
			<td></td>
			<td>Strauss, S. et al., Methods Mol Biol. 2019</td>
		</tr>
		<tr>
			<td>myo-inositol</td>
			<td>Sigma</td>
			<td>I5125</td>
			<td></td>
		</tr>
		<tr>
			<td>N6-benzyladenine</td>
			<td>Sigma</td>
			<td>B3408</td>
			<td></td>
		</tr>
		<tr>
			<td>nicotinic acid</td>
			<td>Sigma</td>
			<td>N4126</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic hinged box</td>
			<td>Electron microscopy sciences</td>
			<td>64312</td>
			<td></td>
		</tr>
		<tr>
			<td>PPM (Plant Preservative Mixture)</td>
			<td>Plant Cell Technology</td>
			<td>PPM</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>pyridoxine hydrochloride</td>
			<td>Sigma</td>
			<td>P9755</td>
			<td></td>
		</tr>
		<tr>
			<td>SURFCUT</td>
			<td></td>
			<td></td>
			<td>Erguvan, O. et al., BMC Biol. 2019</td>
		</tr>
		<tr>
			<td>thiamine hydrochloride</td>
			<td>Sigma</td>
			<td>T4625</td>
			<td></td>
		</tr>
	</tbody>
</table>

live cell imaging of microtubule cytoskeleton and micromechanical manipulation of the arabidopsis shoot apical meristem

了解细胞和组织水平对生长和形态的调控几十年来一直处于生物学研究的最前沿。分子和成像技术的进步使我们能够深入了解生化信号如何影响形态遗传事件。然而，越来越明显的是，除了生化信号，机械线索也影响细胞和组织生长的几个方面。 阿拉伯射枪（SAM） 是一个圆顶形状的结构，负责生成所有地上器官。在植物细胞中培养增生纤维素沉积的皮质微管细胞骨架的组织在空间上是截然不同的。皮质微管模式的可视化和定量评估对于理解SAM细胞的生物物理性质是必要的，因为纤维素是植物细胞壁最坚硬的成分。皮质显微管组织的陈规定型形式也是 SAM 中存在的组织范围物理力的结果。这些物理力的扰动和皮质微管组织的随后监测允许识别参与调解机械感知和转导的候选蛋白质。在这里，我们描述了一个帮助调查此类过程的协议。

图 1显示了从 MBD-GFP 线获得的典型投影图像，其中圆顶中心有包含杂乱无章的皮质微管的细胞，而外围的细胞具有圆周分布（图1A，B），而边界域细胞包含与细胞长轴平行的皮质微管。这些观测显示SAM不同域中皮质微管的空间分布差异。时差成像显示皮质微管对齐从高度无序的阵列到消融后 6 小时内更有条理的阵列...

Watch this Scientific Journal Video about Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem at JoVE.com

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

在这里，我们描述了一个协议，用于在拍摄时皮质微管细胞骨架的活细胞成像，并监测其对物理力变化的反应。

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...

该协议，它帮助我们了解微管细胞骨骼行为是如何发生在植物。同时，它还帮助我们了解这些影响细胞和器官形状的结构成分如何响应机械力的变化。虽然，这种方法要求很少复杂，它提供了强大的，定量的读取后，不同基因型和条件之间的机械响应差异。首先种植阿拉伯多普西斯种子，表达微管结合域与绿色荧光蛋白融合在土壤中，在20至6摄氏度的长日条件下一个星期。发芽后，将幼苗转移到具有足够生长空间的新盆中，使植物生长强劲，并在短日条件下将植物温度在20至16摄氏度。三到五周后，将植物在相同的温度下转移到长日条件下，直到植物螺栓，使花序长到两到五厘米长。对于拍摄阿皮克·梅里斯特姆解剖，放花序，并使用锋利的钳子剥落花底的花，直到很难看到用肉眼去除旧花蕾的花瓣。使用钳子在解剖盘中在农产品中创建一个缝隙，并在厚厚的奥古尔中种植花序底座。将盘子放在解剖显微镜下。从最古老的花蕾开始，用钳子推掉植物的花蕾。当第六至第七阶段的花被移走时，通常会露出来。并使用钳子推下每个花蕾， 直到拍摄阿普蒂克梅里斯特姆是可见的。一旦射光暴露，让新鲜解剖的样品在生长介质中，在乙醇消毒的矩形塑料铰链培养盒与拍摄阿普利斯特姆刚刚暴露在介质表面之上。在培养盒边缘加入几滴无菌去ion化水，并合上盖子以保持包装盒内的湿度。用微孔胶带包裹包装盒子，并在 22 摄氏度的长时间或连续的日条件下放置生长盒 12 至 24 小时。当植物从解剖程序中恢复后，用无菌去维化水覆盖样品，并在解剖显微镜下检查气泡。将培养盒转移到直立共体显微镜阶段，然后选择 40 或 60 倍浸水镜头。将目标下到水中，并检查物体前透镜上的气泡。要去除气泡，请降低舞台，用光学组织轻轻擦拭镜头。然后使用巴斯德移液器将少量水添加到目标的前镜头中，然后再将镜头重新浸入水中。接下来，使用共合显微镜的 GFP 过滤器和光显光模块调整 XY 控制器以定位样品。调整眼部的位置，将射射镜直接放在光源下，沿 Z 轴对焦，直到顶点定位。使用能够刺激的 GFP 的激光照亮样品，并调整显微镜的光学变焦，使整个拍摄阿皮克里斯特姆和舞台一花卉原始在视野中。然后，调整激光输出的功率和增益设置，以获得最佳的噪声比。在允许样品沉淀两到五分钟后，以 0.25 至 0.5 微米 Z 切片间隔获取样品的共合 Z 堆栈，增加约 0.3 微米像素大小分辨率。在采集结束时，立即取出水，将培养盒调回生长室。对于微机械拍摄Apical Meristem扰动，获得皮质微管组织的预消融图像堆栈，正如刚才展示的，并脱雾水从文化盒成文化菜。在解剖显微镜下转移射枪，用干净的0.4乘20毫米注射器针慢慢接近射枪。用针尖短暂接触射针圆顶的外围，确认消融已完成。用无菌的去ion化水重新填充培养皿。然后每毫升碘化丙胺加入10微克，并立即获得图像堆栈，如每两小时演示6小时，返回培养皿到每个时间点之间的孵育。对于数据分析，使用适当的图像分析软件程序生成图像堆栈的表面投影，并使用斐济的 Fibril 工具宏执行皮质微管各向异性的提取。在这里，从微管结合域GP线获得的典型投影图像，在圆顶的中心包含杂乱无章的皮质微管的细胞，在外围的细胞，具有圆周分布和边界域细胞包含皮质微管平行于细胞的长轴对齐。延时成像显示皮质微管对齐方式在消融后六小时内从高度无序的阵列向更有条理的阵列转变。张子可以叠加在皮质微管图像上，提取的信息可以通过绘制一段时间的均等各向异性来表示。在调整激光输出功率和增益设置时，应确保对灯丝进行清晰观察，并尽量避免过度暴露，因为过度饱和信号可能会使拉伸方向在以后的分析中偏向。原子力显微镜也可以进行，以评估机械力的变化实际上如何影响细胞壁的物理状态。

Research

JoVE Journal

Developmental Biology

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Live Confocal Imaging of Developing Arabidopsis Flowers

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Live Cell Imaging of Chromosome Segregation During Mitosis

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

使用纤维素凝块固定 的果蝇 组织用于实时成像

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

细胞细胞成像的微管细胞和微机械操作的阿拉比多普西斯拍摄阿皮克·梅里斯特姆