这项工作是由来自安特卫普大学VA一个博士研究生奖学金的支持;从法兰德斯科学基金会（FWO，11ZI916N），以KS博士奖学金;从FWO（G0D0514N）项目赠款;协调一致的研究活动（GOA）研究经费，从安特卫普大学的研究委员会"叶形态的系统生物学方法";和校际景点波兰人（IUAP VII / 29，MARS），"玉米和拟南芥幼苗和根生长"，从比利时联邦科学政策办公室（BELSPO），以GTSB韩Asard，Bulelani L. Sizani和滨田AbdElgawad都促使视频。

注意:以下协议运动学分析只适用于稳态生长过程中的叶子。此期间的几天6意味着稳定叶伸长率和细胞的长度和在叶扩展空间模式。 1.植物生长和叶片伸长率的措施（LER） <ol><li>选择在稳态增长叶片和感兴趣的发展阶段。 注意:有是稳态生长和重复的增长，这意味着对在同一轴线上的连续叶片类似空间模式之间的差。在幼苗生长的早期阶段，连续的树叶一般的生长区7的尺寸增加变得越来越快所致。虽然有少数较高的叶位置可以有类似的增长模式8，这是可以被接受调查处理的影响过渡阶段。它来比较线路和tr为因此，重要的<html> eatments严格在同一叶位，即使它可能在不同的时间进行开发。即使在恒定伸长率，生长速度轮廓不一定在不同发育阶段是相同的。因此，在相同的发育阶段8，典型地通过出苗后的天数定义为分析叶是重要的。 </li><li>为了在单子叶植物叶片进行增长的全面运动学分析，至少增长15株为每个治疗和基因型生长室控制的条件下。 </li><li>在感兴趣的叶出现（出现从围绕叶片的螺纹）的时间，开始用尺子测量叶的长度每日直至叶完全展开（ 图1i）中 。叶长意味着从土壤水平长度与叶的尖端。小心不要折断或损坏叶，因为这可能会改变其生长。 </li></ol>重1"SRC ="/文件/ ftp_upload / 54887 / 54887fig1.jpg"/&gt; 图1:玉米叶片的运动分析示意图感兴趣的叶子与连续三天来计算叶片伸长率（LER）尺子测量。此后，将叶收获和三厘米段被用于分生组织大小的确定。这是通过测量从基向上的长度，以DAPI染色后，最远端的有丝分裂图进行。 （A）的增殖核分裂和（B）形成核分裂的例子。从中期静脉的另一侧的叶基部第一11厘米用于切割为电池的长度测量十分之一厘米的段。这些测量提供用于创建元长度信息，其用于确定所述成熟细胞长度（L 垫 ）和细胞离开分生组织（L 格 ）的长度的基础。该<eM&gt; LER和L 垫被用于计算所述电池的生产速率（P），而L 的div和L 聚体被用来计算细胞的分生组织（N 聚体 ）的数量。反过来，P和N 聚体被用来计算平均细胞分裂率（D），它是细胞周期的持续时间（T C）的倒数。相同颜色的箭头表示用于计算以下这些箭头的参数的参数。比例尺= 40微米。罗马数字被用来指在协议中所述具体的实验过程。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/54887fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.收获<ol><li>在感兴趣的发育阶段（ 例如，出苗后第三天），选择至少五个代表p从批量lants在其上进行运动学分析。继续测量植物的其余部分如在步骤1.3解释来确定最终的叶长。 </li><li>切割植物的地上部分。为了保持分生部分完好，削减尽可能靠近根部（ 图1II）。 </li><li>从外叶开始，通过由一个轻轻展开逐一去除所有叶到感兴趣的叶子。如果需要的话，从基座移除一些额外毫米至分离的叶子。同时删除受利益（ 图1iii）的叶包围的顶点和小叶子。 </li><li>切断的3厘米的段，从基座上中期静脉的一侧开始，并把它存储在1.5ml试管填充有3:1（体积比）的绝对乙醇:乙酸溶液（小心:戴手套）在4℃放置24小时至数月（ 图1iv）。本段以后将用来确定分生组织的长度。 </li><li>来自静脉的另一侧，切断从基部A 11厘米的段（ 图1ⅴ），并将其放置在一个15毫升管在4℃下填充有无水乙醇为至少6小时，以除去色素（ 图1ⅵ）。 注:后来，仅使用前10厘米测定细胞长度的个人资料（见讨论）。 </li><li>续约另一轮在4℃清洗至少24小时（ 图1vi）的无水乙醇。 </li><li>最后，代替无水乙醇与纯净乳酸（小心:戴手套）用于清洁和存储在4℃下24小时或直至进一步使用（ 图1vi）。 </li></ol> 3.茎尖长度测量<ol><li>制备含有50mM氯化钠（NaCl），5mM的乙二胺四乙酸漂洗缓冲液（EDTA;注意:戴手套）和10mM的Tris（羟甲基）氨基甲烷 - 盐酸（TRIS盐酸; pH7）中。 </li><li>从第2.4节等以3厘米的段阿克它在缓冲器20分钟（ 图1vii）。 </li><li>在等待中，使用漂洗缓冲液，制备的1μg/ ml的4&#39;，6-二脒基-2-苯基吲哚（DAPI）染色溶液，保持在冰上，并在黑暗中。 </li><li>通过将分生组织段在DAPI染色溶液2-5分钟染色细胞核。在冰上并在黑暗中（ 图1vii）工作。 </li><li>通过快速安装在显微镜的玻璃段，并用玻璃盖覆盖它检查荧光信号。表皮细胞应该表现出荧光，而底层的细胞层不应该。 </li><li>如果染色不充分，把段回来为一些额外分钟DAPI染色溶液。 </li><li>要停止染色，在一个显微镜载玻片和盖有盖玻璃清洗缓冲一滴安装段。 </li><li>在放大20倍使用，配有紫外荧光显微镜，允许大约1000表皮的可视化人细胞一次。滚动整个段和寻找增殖核分裂（中期，后期，末期，和胞质分裂），但要避免发展中的气孔（ 图1viii）的形成细胞分裂9。定义哪里最远端的有丝分裂的数字的位置。 </li><li>通过测量叶的基部和最远端表皮有丝分裂图之间的距离确定的分生组织的长度。使用图像分析软件（ 例如，ImageJ的）来测量所述图像帧的全部长度。 <ol><li>算覆盖完整的分生组织的长度（从叶基部到最远端有丝分裂图）的帧的数量和一帧的长度乘以这个号码来得到完整的分生组织的长度（ 图1ix）。 </li></ol></li></ol> 4.池长简介<ol><li>以存储在乳酸（步骤2.5）段和小心将它放置在板凳上。切段机智哈手术刀在1厘米每个（ 图1X）10段。 </li><li>安装在一个显微镜载玻片连续叶段乳酸一小滴。确保始终如一地面向近轴或远轴的一面朝上。原则上，对于一个特定侧没有偏好。 </li><li>使用带有微分干涉对比（DIC）的光学显微镜分析段，从叶基部开始。用图像分析软件测量以一致地选择相同的细胞类型的至少20复制表皮细胞中直接相邻的气孔文件文件的长度。 <ol><li>为此沿各段（每段打住4个位置）的等距位置，并一定要记下每次测量整个叶（ 图1xi）的相应位置。 </li></ol></li><li>通过使用本地多项式平滑p确定在沿叶轴每毫米的平均泡孔长度rocedure，在R-脚本（;补充文件1 图1xii）实现的。 注:R-脚本提供了一系列增加平滑的数据。平滑所需的量是任意的，最好应该只是删除本地噪音，但并不影响整体的曲线。确保使用一个实验内平滑对于所有样品的相同量。 </li><li>平均在植物之间的各位置的细胞的长度，并计算标准误差来创建沿着叶片轴线的细胞长度轮廓。 </li></ol> 5.运动参数计算（见补充文件2） <ol><li>通过取叶片长度的变化的两个连续时间点之间（ 例如，24小时，如在步骤1.3）和由时间间隔除以计算LER。 </li><li>计算对应于从基部远端的位置，其中的细胞达到成熟CE的95％的增长区（L GZ）的长度在平滑细胞长度轮廓LL长度。 <ol><li>取为平均以下该位置（ 图2）中的所有细胞的长度的平滑信元长度信息95％的各位置。 </li><li>比较平滑的细胞长度（步骤4.4）在各位置计算出的95％的细胞长度。 （;见补充数据2 图2）从叶子的基部开始，生长区在其中电池实际长度等于以下细胞长度的95％的位置结束。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/54887/54887fig2.jpg" /> 图2:确定生长区的端分生组织:在用红色星所指示的位置时，电池实际尺寸小于95％以下的该位置的所有小区的平均泡孔大小的（红色虚线）（红色固体线）。在生长区（L GZ的结束;具有AB表示泰伦星）位于其中，95％以下这个位置（蓝色实线的所有单元的平均单元尺寸（蓝色虚线））等于实际单元格大小。除法区（D）中，伸长区（E），和成熟区（M）。虚线箭头表示当地的规模和平均规模上，从基础岗位叶的尖端移动时叶的远端部分的95％之间的衔接。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/54887fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>计算伸长区（L EL）作为生长区（L GZ）和分生组织大小的长度之间的差的长度（L 聚体 ;在第3步确定）。 </li><li>计算出的成熟细胞的长度（L 垫 ）作为成熟区中的平均细胞长度。 </li><li>用L 垫除以LER获得电池生产速率（P）。 </li><li>计算伸长区（N EL）为N 的gz和N 聚体之间的差异细胞数。在分生组织（N- 聚体 ）的细胞的数目等于设在对应于所述分生组织的间隔单元的累计数。在生长区（N GZ）的细胞的数目等于位于对应于增长区中的间隔单元的累计数。 </li><li>计算平均细胞分裂率（D）为P / N 聚体 。细胞周期的持续时间（T C）等于LN（2）/ D。 </li><li>除以Ñ 埃尔用P计算伸长区（T EL）的时间。在划分时区的时间等于日志2（N MER）* T C。细胞离开分生组织的长度（L 格 ）等于从平滑单元长度信息在分生组织的端部的细胞长度。 </li><li>计算使用下列公式单元格平均扩张速度（R EL）:LN（L 垫 ）-ln（L DIV）] / T EL。 </li></ol>

作者宣称，他们没有竞争的经济利益。

在玉米叶片完整运动学分析，使叶片生长的细胞基础的决心，并允许有效采样战略的设计。虽然该协议是相对简单的，一些谨慎以下述关键步骤，建议:（1）它不破坏分生组织分离年轻的，封闭的叶（步骤2.3）中，由于分生组织长度判定（步骤3）是重要的，需要的完整分生组织到场。事先可能需要一些练习。 （2）分生长度确定是基于的核分裂解释。因此，我们建议，一个实验中，同一个人，由于执行人进行这些测量，以减少偏差。 （3）虽然只有十厘米用于电池实际长度的测量，其包括11厘米收获细胞长度测量（步骤2.5）的链段时，由于段的边缘确实没有重要的是并不总会完全浸没在乙醇和/或乳酸。因此，这种额外厘米确保生长区的前十个厘米结算。 （4）最后，它要检查所有运动参数的结果，以确认它们是正确有用的。在表3中 ，例如，在干旱胁迫植物的细胞生产速率（P）由71％下降，或者，换句话说，P 干旱是P 控制的0.29。由于电池生产取决于平均细胞分裂速率（D; -63％），并在分生组织细胞的数量（N 聚体 ; -20％; 图3），以下可用于验证输出P（0.29）≈ ñ 滨海 （0.8）×D（0.37）。同样可以应用于LER = P×1 垫 。 根据可用的设施，该协议可以在一些步骤进行修改。 （1）多日常手册叶长度测量（步骤1.3）先进适用的替代是根据线速度位移传感器（LVDT传感器）13。其优点是，它使测量在更高的时间分辨率，范围从几分钟到秒。 （2），这是至关重要包括采样期间完全生长区。然而，环境条件，基因型，品种，并检查了发育阶段可能改变的生长区的长度。因此，它是有益的，使生长区的长度的一个第一估计响应于实验治疗。该协议是基于自交系B73增长在25°C / 18°C（昼/夜）在300-400μEm-2 S -1（光合有效辐射- PAR）。 （3）为了形象化核分裂（步骤3.8），而无需使用荧光，一Fuelgen污点可以应用14。 （4）数据的平滑化也可以在MS Excel代替中的R用五点之四完成dratic与"LINEST"函数拟合15。 这种技术的一个重要的限制是，整个生长区需要稳态生长期间收获。为了确保稳定状态生长，植物必须理想地在受控生长条件下生长的，因为在光强度，温度或湿度波动影响叶伸长率16。由于这个原因，运动分析是不太适用于田间条件下生长的植物。另一个缺点是，需要的叶子被显现，这使得不可能以检查仍然由老叶包围叶。最后，该协议适用于大增长区单子叶植物品种涵盖至少几厘米。在较小的单子叶植物（ 例如，早熟禾 ，大米）运动学分析，但是，是可行的，但是在样品制备17,18需要进行一些修改。 另一个经常使用ð方法来分析不同的生长表型是古典增长分析。此分析量化全株相对生长率，并且确定光合衍生碳的基本分配到一个新的光合面积和进厂19的其他部分。运动学分析，但是，允许对被耦合到根据量化细胞分裂和扩张的生长表型的底层细胞过程的更详细的了解。此外，为了确定每个生长区的长度的可能性允许沿叶轴分子测量的到由所研究的治疗影响的特定发育阶段的相关性。这是为数据解释关键。当在时间或预算或感兴趣的特定发育阶段时的限制，此分析的结果允许聚焦采样，减少分析所需的样本数。总之，运动学分析启用■在叶生长应激反应和遗传变异细胞基本参数的确定，可以将它们链接到上游的监管程序（ 表2）。

生长分析依赖于一组工具通常使用由植物科学家描述基因型确定生长的差异和/或表型响应于环境因素。它们包括全厂的大小和重量测量或器官和增长率的计算来探讨经济增长的内在机制。器官发育是细胞分裂和扩大在细胞水平确定。因此，包括在生长分析这两个过程的量化的关键是理解在整个器官1生长的差异。因此，关键是要具有适当的方法来确定细胞生长参数是相对容易由非专业的实验室使用。 运动学分析已经被确立为一种方法提供的器官发育模型2发展的一个强大的框架。这项技术对线性系统进行了优化，如拟南芥根和单子叶叶子，也为非线性的系统中，例如双子叶叶片3。如今，这种方法被越来越多地用于研究如何遗传，激素，发育和环境因素影响细胞的分裂和各种器官的扩展（ 表1）。此外，它也提供了一个框架，以细胞过程链接到他们的潜在生化，分子和生理规章（ 表2），尽管限制可以通过器官大小和空间组织为需要更高量的植物材料的技术（ 例如，代谢物施加测量，蛋白质组学， 等等 ）。 单子叶叶子，如玉米（ 玉蜀黍 ）叶，表示其中的细胞从朝向尖叶的基部移动线性系统，通过分生组织和伸长区顺序地传递到到达成熟区。这使得它的增长4的空间格局定量研究的理想模式系统。此外，玉米叶有较大的增长区域（分生组织和伸长区跨越几厘米5），并在其他组织层次的研究提供了可能性。这允许（假定）的监管机制控制细胞分裂和扩大，通过一系列的分子生物学技术，生理测量和细胞生物学的方法（ 见表2）通过运动学分析定量调查。 在这里，我们提供了在单子叶树叶进行运动分析的协议。首先，我们将解释如何进行这两种细胞分裂和细胞伸长的正确分析，沿叶轴位置以及如何计算运动学参数的函数。其次，我们还显示如何可以用作用于取样设计的基础。在这里，我们讨论了两种情况:高分辨率的采样ð集中采样，从而提高了数据的解释和时间/钱储蓄，分别为。 表1.运动概述分析了细胞分裂和各种器官的扩张量化方法。 <table border="1" fo:keep-together.within-page="1"><tbody><tr><td> 器官 </td><td> 参考 </td></tr><tr><td>单子叶植物的叶子</td><td> 16，20，21，22 </td></tr><tr><td>根尖</td><td> 2，23，24，25，26，27，28，29 </td></tr><tr><td>双子叶植物的叶子</td><td> 21，30，31 </td></tr><tr><td>拍摄顶端分生组织</td><td> 32 </td></tr></tbody></table> 表1.运动概述分析了细胞分裂和各种器官的扩张量化方法。 表2.由运动分析来在分子水平上的调控量化细胞过程之间的链接。参考资料细胞过程的量化链接到来自于不同物种和器官生物化学和分子分析结果的各种研究。木聚糖endotransglucosylase（XET），丙二醛（MDA），细胞周期蛋白依赖性激酶（CDK）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/54887table2large.jpg" target="_blank">请点击这里查看此表的放大版本。</a>

<table><tbody><tr><td>Pots</td><td>Any</td><td>Any</td><td>We use pots with the following measures, but can be different depending on the treatment/study: bottom diameter: 11 cm, opening diameter: 15 cm, height: 12 cm. We grow one maize plant per pot.</td></tr><tr><td>Planting substrate</td><td>Any</td><td>Any</td><td>We use potting medium (Jiffy, The Netherlands), but other substrates can be used, depending on treatment/study.</td></tr><tr><td>Ruler</td><td>Any</td><td>Any</td><td>An extension ruler that covers at least 1.5 meters is needed to measure the final leaf length of the plants.</td></tr><tr><td>Seeds</td><td>Any</td><td>NA</td><td>Seeds can be ordered from a breeder.</td></tr><tr><td>Scalpel</td><td>Any</td><td>Any</td><td>The scalpel is used during leaf harvesting to detach the leaf of interest from its surrounding leaves and right after harvesting to cut a proper sample for cell length and meristem length measurements.&amp;nbsp;</td></tr><tr><td>15 mL falcon tubes</td><td>Any</td><td>Any</td><td>The 15 mL falcon tubes are used for storing samples used for cell length measurements during sample clearing with absolute ethanol and lactic acid.</td></tr><tr><td>Eppendorf tubes</td><td>Any</td><td>Any</td><td>The eppendorf tubes are used for storing samples used for meristem length measurements in ethanol:acetic acid 3:1 (v:v) solution.</td></tr><tr><td>Gloves</td><td>Any</td><td>Any</td><td>Latex gloves, which protect against corrosive reagents.</td></tr><tr><td>Acetic acid</td><td>Any</td><td>Any</td><td>CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1; Flammable liquids, categories 1,2,3</td></tr><tr><td>Absolute ethanol</td><td>Any</td><td>Any</td><td>CAUTION: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (permeator), of ingestion</td></tr><tr><td>Lactic acid&amp;nbsp;&gt;98%</td><td>Any</td><td>Any</td><td>CAUTION: Corrosive to metals, category 1 Skin corrosion, categories 1A,1B,1C Serious eye damage, category 1</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Any</td><td>Any</td><td>CAUTION: Acute toxicity (oral, dermal, inhalation), category 4 Skin irritation, category 2 Eye irritation, category 2 Skin sensitisation, category 1 Specific Target Organ Toxicity &amp;ndash; Single exposure, category 3</td></tr><tr><td>Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)</td><td>Any</td><td>Any</td><td>This material can be an irritant, contact with eyes and skin should be avoided. Inhalation of dust may be irritating to the respiratory tract.</td></tr><tr><td>4&amp;prime;,6-Diamidine-2&amp;prime;-phenylindole dihydrochloride (DAPI)</td><td>Any</td><td>Any</td><td>Cell permeable fluorescent minor groove-binding probe for DNA. Causes skin irritation. May cause an allergic skin reaction. May cause respiratory irritation.</td></tr><tr><td>Ice</td><td>Any</td><td>NA</td><td>The DAPI solution has to be kept on ice.</td></tr><tr><td>Fluorescent microscope</td><td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td><td></td><td>Any fluorescent microscope can be used for determining meristem length.</td></tr><tr><td>Microscopic slide</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Cover glass</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Tweezers</td><td>Any</td><td>Any</td><td>Tweezers are needed for unfolding the rolled maize leaf right after harvesting in order to cut a proper sample for cell length and meristem length measurements.&amp;nbsp;</td></tr><tr><td>Image-analysis software</td><td>Axiovision (Release 4.8) from Zeiss</td><td>NA</td><td>The software can be downloaded at: http://www.zeiss.com/microscopy/en_de/downloads/axiovision.html. Other softwares such as ImageJ (https://imagej.nih.gov/ij/) could be used as well.</td></tr><tr><td>Microscope equipped with DIC</td><td>AxioScope A1, Axiocam ICm1 from Zeiss or other</td><td></td><td>Any microscope, equipped with differential interference contrast (DIC) can be used to measure cell lengths.</td></tr><tr><td>R statistical analysis software</td><td>R Foundation for Statistical Computing</td><td>NA</td><td>Open source; Could be downloaded at https://www.r-project.org/</td></tr><tr><td>R script</td><td>NA</td><td>NA</td><td>We use the kernel smoothing function locpoly of the Kern Smooth package (Wand MP, Jones MC.&amp;nbsp; Kernel Smoothing: Chapman &amp;amp; Hall/CRC (1995)). The script is available for Mac and Windows upon inquiry with the corresponding author.</td></tr></tbody></table>

kinematic analysis of cell division and expansion: quantifying the cellular basis of growth and sampling developmental zones in zea mays leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

在这里，我们将展示他们的叶子增长方面遭受干旱胁迫条件（干旱，34％SWC）精心浇灌植物（控制，54％的土壤含水量（SWC））和植物之间的比较。所有的植物在生长室生长控制的条件下（16小时白天/ 8小时，夜幕下，25℃/ 18℃，昼/夜，300-400μEm-2秒-1光合有效辐射（PAR）的干旱情况，直至正确的SWC达到再进一步保持代扣水建立的。在初步研究中，它被定义的第五叶出现这种胁迫条件下制定的第一个。因此，被选为叶干旱胁迫植物的兴趣。最终的叶长度（LL）比充分浇水的植物，这是由于较低LER（ 表3）中的一个较低的40％。要理解LEA的蜂窝基础˚F生长反应，我们进行了运动学分析。总体而言，叶伸长率是电池生产中的分生组织和在伸长区确定成熟细胞大小（ 图3）的功能。因此，叶的缩短必定是由于在一个或两个这些参数的影响。我们的结果表明，虽然成熟细胞的长度（L 垫 ）没有受到影响，在电池的生产速率（P）减少了71％（ 表3）;● 垫取决于离开分生组织细胞的长度（L 格 ）中，时间这些细胞在伸长区（T EL）花，相对细胞扩张速率（R EL）。观察对于L 分区没有任何影响，，且R EL是在干旱条件下减少了67％。这种效果是由几乎三倍Ť 埃尔 ，这导致在相同的升 垫的补偿&lt;/子&gt;如在控制的情况。在电池的生产速率的减少，反过来，引起两个较低的平均细胞分裂率（D）和在分生组织（N 聚体 ）更小数目的细胞。分生组织（L 聚体 ）的长度相较于生长区（L GZ），这是不显著影响的长度减少。总之，这些结果表明，在这种情况下，细胞数，与细胞大小，不得不在确定叶片大小主要作用。 <table border="1" fo:keep-together.within-page="1"><tbody><tr><td> 运动学参数 </td><td> C </td><td> ð </td><td> ％变动CD </td><td> p值 </td></tr><tr><td>二</td><td> 727±15 </td><td> 436±23 </td><td> -40 </td><td> 0.000 </td></tr><tr><td> LER（毫米/小时） </td><td> 2.8±0.1 </td><td> 0.8±0.0 </td><td> -73 </td><td> 0.000 </td></tr><tr><td>升垫 （微米） </td><td> 140±3 </td><td> 130±6 </td><td> -7 </td><td> NS </td></tr><tr><td> P（细胞/小时） </td><td> 20±1 </td><td> 6±0 </td><td> -71 </td><td> 0.000 </td></tr><tr><td> D（细胞·细胞-1·小时-1） </td><td> 0.028±0.003 </td><td> 0.010±0.001 </td><td> -63 </td><td> 0.000 </td></tr><tr><td> T C（小时） </td><td> 26±3 </td><td> 71±7 </td><td> +173 </td><td> 0.000 </td></tr><tr><td> ŧ 格 （HR） </td><td> 249±27 </td><td> 656±71 </td><td> +164 </td><td> 0.000 </td></tr><tr><td>大号聚体 （毫米） </td><td> 15±1 </td><td> 11±1 </td><td> -25 </td><td> 0.010 </td></tr><tr><td> 滨海 ñ </td><td> 740±31 </td><td> 590 + 20 </td><td> -20 </td><td> 0.000 </td></tr><tr><td> - [R EL（微米·微米<suP&gt; -1·小时-1） </td><td> 0.043±0.002 </td><td> 0.014±0.001 </td><td> -67 </td><td> 0.000 </td></tr><tr><td>升格 （微米） </td><td> 24±2 </td><td> 23±2 </td><td> -4 </td><td> NS </td></tr><tr><td> ŧEL（HR） </td><td> 42±2 </td><td> 125±11 </td><td> +199 </td><td> 0.000 </td></tr><tr><td> ñ 埃尔 </td><td> 844±54 </td><td> 735±65 </td><td> -13 </td><td> NS </td></tr><tr><td> L G Z（毫米） </td><td> 68±1 </td><td> 61±5 </td><td> -11 </td><td> NS </td></tr><tr><td> N G - ž </td><td> 1584±33 </td><td> 1326±66 </td><td> -16 </td><td> 0.010 </td></tr></tbody></table> 第五叶的稳态增长的过程中细胞分裂和细胞扩张 表3. 运动分析。经受干旱胁迫数据水分充足的植物和植物的平均值±SE（N = 5，对于LH:N = 10）。学生t检验用作统计分析。参数:叶长度（LL），叶伸长率（LER），成熟细胞的长度（L 垫 ），电池的生产速率（P），细胞分裂速率（D）中 ，细胞周期持续时间（T c）中 ，时间在分割区（ Ť 格 ），分生组织的长度（L 聚体 ），细胞在分生组织（N- 聚体数），相对细胞伸长速率（R EL），而使分生组织（L 格的细胞）的长度，时间在伸长区（T EL），在伸长区（N EL）中，生长区的长度（L GZ），在生长区细胞的数目（N GZ），水分充足条件（C）中，干旱胁迫的细胞数（ D），一<html> □不显著（NS）。 <img alt="图3" src="/files/ftp_upload/54887/54887fig3.jpg" /> 图 3: 在运动学参数之间的关系的最后叶长（LL）依赖于叶片伸长率（LER），这反过来又依赖于电池生产性（P）和成熟细胞的长度（L 垫 ）。在分生组织（N- 聚体 ）的细胞的数量和细胞分裂率（D）的共同决定P，细胞周期持续时间（T c）中D的倒数。在伸长区（T EL），长度细胞离开分生组织（L 格 ）的，相对细胞扩张速率（R EL）时间确定成熟细胞的长度（L 垫 ）。在伸长区（N EL）细胞的数量。.jove.com /文件/ ftp_upload / 54887 / 54887fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 除了其作为器官生长的详细分析应用程序，运动学分析提供了一幅图叶片生长区的定位，并允许以带下分辨率抽样分子和生化分析4,10,11。作为一个例子， 图4A示出了沿玉米叶中如上述进行同样的条件下的植物的生长区丙二醛（MDA）的定量。 MDA是由活性氧的脂质过氧化过程中形成的次级代谢产物，反映了氧化损伤12的水平。 MDA含量在叶的每一厘米测量，从它的基础，这使得沿生长梯度11浓度变化的分析开始。作为干旱处理造成40％的叶缩短，发育区的长度（梅里干，对照植株伸长区，成熟的区）没有匹配相同的区域在干旱强调植物（ 图4B）的长度。如在图4B中看到的，在对照植物的分生组织是大约1.5厘米长，和伸长区从叶基1.5〜6.8厘米跨越。在强调植物，分生组织是从0至1.1厘米，1.1至6.1厘米伸长区本地化。 MDA水平和整个生长区在干旱胁迫的植物与对照相比，治疗均升高。与运动学分析的帮助下，我们可以得出结论，MDA含量在控制和治疗干旱成熟区特别增加。在不知道的发展区的大小，我们可以得出结论的是，在干旱胁迫下，在较早的发育阶段的MDA含量的增加比在对照植物，这将是错误的，因为它没有考虑到由于该区域的移动到t他叶缩短。因此，之前的生化指标进行运动分析是至关重要的，以便正确地解释数据。 <img alt="图4" src="/files/ftp_upload/54887/54887fig4.jpg" /> 图4: 高分辨率生化测定实施例和采样为聚焦分析 （A）的丙二醛（MDA）的沿玉米叶生长区（分成十段）测定植物的浓度生长在充分浇水条件和植物经受干旱。治疗10。数据是平均值±SE（N = 5）。双向ANOVA用作统计分析，并且p值旁边的图面板（因素:干旱（d）和链段（多个））呈现; FW:鲜重。在控制和干旱强调植物的不同生长区（B）可视化。除法区（D）中，伸长区（E）和成熟区域（M）。细胞被放大了125倍。 （ 三 ）控制和干旱处理的植物细胞长度的轮廓。数据是平均值±SE（N = 5）。在聚焦取样（D）的差异控制和干旱处理的植株。蓝色框表示采样位置比较同一发展阶段的两种治疗。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/54887fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 除了提供比较高分辨率研究相同发育区的可能性，该运动学分析也可以被用作用于更集中的分析，其中，采取只含在相同发育阶段的细胞的特定区域的样本的基础。我们比较了细胞长度型材，由微观细胞的长度测量的平滑提供，在第一10个C从控制和干旱胁迫的植物的叶基entimeters 图4C示出的是，使用细胞长度信息，我们可以判断发育区的长度，也可以按照在对照和干旱胁迫植物的生长速率动力学。这使得在分生组织增殖细胞的特定的抽样（前急剧增加），扩大中的细胞伸长区（在陡峭的增加的中间），并在成熟区年轻成熟细胞（高原开头）。我们的研究结果表明，由于响应于干旱叶缩短，这些区域没有在控制和在胁迫植物对应。为了样增生，扩张，并在控制植物年轻的成熟细胞，我们需要的第一，第五和第九从叶基厘米，分别为。但是，在强调的植物，这些段分别对应于第一，第四和第八厘米。这种更集中的抽样已被证明是在更费力，费钱，或费时的研究，如RNA测序，蛋白质组学，代谢等的情况下是有用 补充文件1:R-脚本多项式平滑程序 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/Supplementary_Data_1.docx" target="_blank">请右键点击下载此文件。</a> 补充文件2:运动参数的计算 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54887/Supplementary_Data_2.xlsx" target="_blank">请右键点击下载此文件。</a>

Watch this Scientific Journal Video about Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves at JoVE.com

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础<em&gt;玉米</em&gt;叶

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

kinematic-analysis-cell-division-expansion-quantifying-cellular-basis

Universidad San Sebastian

Research

JoVE Journal

Biology

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

10.8K 次观看.  University of Antwerp. Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies. 

视频: Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

科研

生物化学

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

发育生物学

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

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 ...

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in indentation mode, we show how to measure an elastic modulus and use it to evaluate cell wall mechanical properties. In addition, we also explain how to evaluate turgor pressure. The main advantages of atomic force microscopy are that it is non-invasive, relatively rapid (5~20 min), and that virtually any type of living plant tissue that is superficially flat can be analyzed without the need for treatment. The resolution can be very good, depending on the tip size and on the number of measurements per unit area. One limitation of this method is that it only gives direct access to the superficial cell layer.

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

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

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

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

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

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 ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

生物工程

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

生物学

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

细胞扩散过程中细胞边缘动力学的定量分析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础

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观察有丝分裂和动力学在现场斑马鱼胚胎

不同植物种的射击顶端共聚焦活图

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

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

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

从长期来看，高解析度激光共聚焦时间流逝成像

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

细胞扩散过程中细胞边缘动力学的定量分析

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础

摘要

探索更多视频

此视频中的章节

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

观察有丝分裂和动力学在现场斑马鱼胚胎

不同植物种的射击顶端共聚焦活图

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

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

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

从长期来看，高解析度激光共聚焦时间流逝成像

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

细胞扩散过程中细胞边缘动力学的定量分析

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