Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

这项工作得到了康诺特基金新调查员奖、加拿大创新基金会、NSERC发现赠款计划（授予RGPIN-2015-05114和RGPIN-2020-05881）、曼彻斯特大学和多伦多大学联合研究基金以及多伦多大学XSeed项目的支持。

1. 细胞播种
注：描述的细胞扩散协议使用小鼠胚胎成纤维细胞（MEF）执行，表达PH-Akt-GFP（PIP3/PI（3，4）P 2的荧光标记）。该细胞系通过CRISPR介质基因编辑，通过基因学整合PH-Akt-GFP（添加基因#21218）的表达结构而生成。然而，其他在基因组中短暂表达或集成的荧光标记也可以用于此测定。为了实现最佳图像分割，我们建议使用细胞质均匀分布的荧光标记，例...

<ol>
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描述的细胞扩散检测允许连续跟踪形态变化（如细胞大小和形状）和细胞边缘运动（即突起速度和缩回频率），这是大多数细胞扩散协议19，24中缺少的功能。虽然常用的端点细胞扩散检测允许确定细胞扩散速度，但这些测定无法解决细胞边缘运动的时间动力学。缺乏时间信息限制了检测和量化跛脚突起-缩回周期变化的能力。
<p class="jov...

拉梅利波德突起是生长细胞前部形成的突出细胞骨结构。在lamilipodia，在Arp2/3复合物的帮助下，行为物的聚合和福明斯创造了一个快速生长的分支行为网格，推动对等离子膜1，2。网格工程产生的推动力在物理上推动细胞向前推进1，3，4，5。Arp2/3复杂或信号通路的耗竭，对于跛脚突出至关重要，通常会损害细胞迁移6，7。虽然跛脚足缺细胞的迁移也已报告8，9，跛脚足在细胞迁移中的重要性是显而易见的，因为这种突起结构的耗竭扰动细胞通过复杂的生物微环境移动的能力6，10。
理解迁移细胞中跛脚足的调控的一个主要障碍是跛脚突出动力学的自然变异性、大小和形状11、12、13、14。此外，最近的研究表明，跛脚皮迪亚表现出复杂的突出行为，包括波动，周期性，加速突出14，15。与迁移细胞6、16的高度可变的跛脚足症相比，在细胞扩散过程中形成的跛脚足足12更为均匀。由于扩散和迁移细胞的突起活动是由相同的大分子组件驱动的，包括分支作用网络、收缩性活体蛋白束和基于内格林的细胞基质粘附17、18，因此扩散细胞被广泛用作研究跛脚体动力学调控的模型。
细胞扩散是一个动态的机械化学过程，悬浮中的细胞首先通过基于内格林的附着力17、19、20粘附粘附，然后通过延长基于作用的突起21、22、23进行扩散。在扩散阶段，从细胞体中喷出的跛脚足部突出异位和持久，很少或根本没有收回或停滞12。最常用的细胞扩散协议是端点检测，其中扩散细胞在电镀19，24后在不同时间固定。这些检测虽然快速而简单，但检测跛脚皮迪亚动态特征的变化的诊断能力有限。为了确定控制跛脚足动力学的分子机制，Sheetz小组率先对活扩散细胞进行了定量分析，并发现了细胞边缘突起11、12、22的许多基本特性。这些研究表明，活细胞传播检测是细胞生物学实验室工具箱中一种强大而有力的技术。尽管如此，细胞生物学界目前还无法为活细胞传播检测提供详细的协议和开源计算工具。为此，我们的协议概述了成像活传播细胞的程序，并提供了一个自动化的图像分析工具。为了验证这种方法，我们使用Arp2/3抑制作为实验治疗，并表明抑制Arp2/3复合体的功能不会阻止细胞扩散，但会导致细胞突起速度显著降低，以及细胞边缘突起的稳定性，从而产生锯齿状细胞边缘。这些数据表明，活细胞成像和自动图像分析的结合是分析细胞边缘动力学和识别调节跛脚皮迪亚的分子组件的有用工具。

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		<tr>
			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red</td>
			<td>Wisent Bioproducts</td>
			<td>319-005 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil</td>
			<td>Cargille</td>
			<td>16241</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution (200 mM)</td>
			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
			<td>VWR</td>
			<td>CA48366-227-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Body, Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
			<td>Nikon</td>
			<td>MRD01605</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk, Crest Light V2</td>
			<td>CrestOptics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, Style 2</td>
			<td>Electron Microscopy Sciences</td>
			<td>78326-42</td>
			<td></td>
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</table>

quantitative analysis of cell edge dynamics during cell spreading

细胞扩散是一个动态过程，其中悬浮在介质中的细胞附着在基材上，并把自己从圆形压扁成薄而分散的形状。在细胞基底附着物之后，细胞形成一块从细胞体中散发出的薄薄的跛脚状。在lamellipodia中，球状作用素（G-actin）单体聚合成密集的丝状活性蛋白（F-actin）网状物，推动对等离子膜，从而提供细胞扩散所需的机械力。值得注意的是，控制跛脚细胞聚合的分子分子对许多其他细胞过程至关重要，如细胞迁移和内分泌。 
由于扩散细胞形成横跨整个细胞外围并持续向外扩张的连续跛脚状体，细胞扩散检测已成为评估跛脚突起动力学的有效工具。虽然已经制定了细胞传播检测的若干技术实施，但目前缺乏对工作流程的详细描述，其中包括分步协议和数据分析的计算工具。在这里，我们描述了细胞扩散检测的实验过程，并提出了一个开源工具，用于定量和公正地分析细胞边缘动力学在扩散过程中。当与药理学操作和/或基因沉默技术相结合时，此协议可适应调节跛脚突出的分子参与者的大规模屏幕。

上述协议描述了扩散细胞活细胞成像的实验程序，以及用于细胞扩散动力学定量分析的计算工具。计算工具可以采用低通量或高通量格式，以识别调节细胞前沿作用聚合机械的分子玩家。
实验程序的示意图表示在图1中描述。细胞扩散检测是在不朽的小鼠胚胎成纤维细胞上进行的，稳定地表达了标有eGFP25的Akt蛋白激酶的丛斯特林同源（P...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

在此协议中，我们介绍了基于活细胞显微镜的细胞扩散检测的实验程序。我们为荧光标记细胞的无偏见分割和细胞扩散过程中的跛脚足动力学定量分析提供了开源计算工具。

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 ...

本视频中描述的协议将定义传播细胞的真人大小成像所需的实验程序，以及使用计算工具，以公正和自动化的方式量化传播动态的细胞。细胞扩散资产允许在细胞扩散过程中持续跟踪细胞边缘运动和形态变化，这是大多数现有细胞扩散检测中缺失的一个特征。同时，本协议还实现了计算机自动处理和分析的使用，提供了关于细胞循环、区域和突出缩还周期的信息。这种自动处理减少了数据分析中的偏差，并为大量细胞提供了分析细胞传播动力学的可靠方法。当与药物治疗或基因科学和技术相结合时，此协议可适应调节细胞突起的分子参与者的大规模速度。对于给定协议，使用的细胞是小鼠胚胎成纤维细胞，通过基因编码PH-Akt-GFP，允许荧光标记等离子膜。在细胞展开检测之前，培养一盘细胞达到90%的共性。一旦细胞达到适当的对流，火焰22乘22毫米覆盖滑动，并将其放入35毫米细胞培养盘。涂上在PBS中稀释的纤维素，使盖片的浓度为每毫升2.5微克。将带有盖子的盘子放入 37 度孵化器中一小时。一小时后，吸气纤维素，确保不要触摸与移液器尖端的盖滑。用PBS洗碗，轻轻在盖子周围吹两到三次。对于细胞播种，首先从细胞盘中吸气细胞培养介质。之后，用温暖的PBS洗碗。在菜中加入 650 微升的 trypsin EDTA，使菜向倾斜，以均匀地分配酶。将带试剂的菜放入孵化器中一分钟。孵化后，您首先需要将 10 毫升细胞培养介质添加到离心管中。接下来，快速在菜中加入另外10毫升的介质，以淬灭试穿素。为了稀释将播种到盖滑上的细胞，将一毫升的菜肴内容放入离心管中。从管子上，移液器约500至1000微升稀释细胞进入菜中，盖滑。确保盖滑在 10% 的汇流或每毫升 5 万个细胞，并根据需要调整稀释细胞的体积。这些细胞的目的是在图像采集过程中建立一个焦点平面。在盘中剩余的细胞中，每次处理将五分之一的细胞放入一个小盘子中。这些细胞将被分析为传播动力学。将通道盘和盖滑盘放入孵化器中 8 至 24 小时。为了测试Arp2/3对细胞传播的重要性，首先在控制和治疗离心管中加入5毫升的细胞培养介质。接下来，在两个较大的离心管中各加入20毫升缺乏酚红色的 dmem。派珀特要么药物CK-666，这是Arp2/3复合物的抑制剂或控制治疗，如DMSO到大小管。要开始对细胞进行药理治疗，取出在一夜之间孵育的通道盘。从所有菜肴中吸气细胞培养介质，用温暖的 PBS 洗碗。将含有 CK-666 或控制补充介质的小管放入每个通道盘中，并将相关管子的内容添加到每个通道中。给每道菜贴上正确的药物治疗标签，然后将菜肴放回孵化器中一小时后再孵化一小时，从每道菜中吸气补充药物介质。接下来，在所有菜肴中加入温暖的 PBS，以便彻底去除所有剩余的酚红色介质。加入230微升的特普辛EDTA，将菜肴放入孵化器中一分钟。从孵化器中取出试剂，然后将缺乏苯酚红的药物补充的五毫升加入指定为管 B 的管中，然后在相关菜中再加入五毫升相同介质，以淬火。将介质上下多次，以便将所有细胞从盘子中分离。将菜的所有内容转移到另一个已被指定为管子的管子中，A.In 准备适合成像的细胞稀释，将一毫升细胞从A管转移到B管中。重复每次治疗的所有稀释步骤。将所有管子放入孵化器中45分钟，使细胞从尝试性化中恢复。确保可容纳 22 乘 22 毫米盖滑的细胞磁室的所有部分在使用前都经过彻底清洁。用盖子从孵化器中取出盘子。吸气细胞培养介质，用温暖的 PBS 清洗封面滑。使用一对钳子取下盖滑，轻轻地将盖滑倒放在磁室的底部板上。接下来拿起硅胶垫片，放在盖滑的顶部。将磁室的主体连接到底部板上。加入一毫升缺乏酚红色的 dmem，与磁室的相关处理相对应。采取无绒组织，并仔细擦在主体和底部板之间的外壳，以检查任何泄漏。要完成磁室，将透明盖降低到主体上。最后用喷水的纸巾擦拭盖底，然后用70%乙醇喷洒另一块。将共聚焦显微镜的舞台顶部孵化器预热至 37 度。将磁室放在目标顶部。在获取扩散动力学之前，将焦点放在绿色荧光通道中已经两极分化的细胞上。这确保了扩散细胞在连接到盖滑时成为焦点。从从孵化器中取出的管 B 中取出磁室和移液器 500 微升的透明盖。将透明盖重新放在顶部。为了识别适合细胞扩散分析的细胞，搜索绿色光环，这些光环代表尚未附着在盖滑上的细胞或处于附着的最初阶段的细胞。获取图像并保存文件。完成细胞传播的图像采集后，首先运行兼容的 Python IDE（如 Spyder）。对于细胞传播动力学的计算分析，定位并打开相关脚本包中提供的细胞传播GUI文件。按下将打开后台分析GUI面板的运行按钮。GUI 提供分析所需的所有设置。要分析细胞区域和循环，请输入采集文件和细胞传播区域选项卡中的必要设置。必须根据单元格类型和图像采集参数进行调整。按下运行后，脚本将为所有分布的单元创建细胞循环和区域与时间图。有关基图数据，按下基图生成器和分析选项卡。此分析要求裁剪输入文件单单元格图像堆栈。在插入所有设置并按下运行后，脚本将为给定单元格创建多个基图。左边是来自DMSO和CK-666治疗的代表性细胞，使用所提供的脚本自动分割。与对照细胞所表现出的同源和高度圆形形态相反，Arp2/3抑制细胞的循环性降低。此外，自动生成的基图提供时间分辨率，对于理解突起的动态至关重要。控制细胞持续突出，几乎没有收回。相比之下，Arp2/3 抑制会破坏在整个传播过程中缩还事件增加所表明的突出的稳定性。这些视频应为您提供如何正确播种细胞、执行药理治疗以及准备细胞传播动力学量化所需的成像和计算工具的理解。此协议可与细胞球菌荧光成像和迁移测定进一步结合，以确定控制细胞突起的分子参与者。谢谢你的观察和祝你的实验好运。

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

科研

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

确定在细胞培养的细胞数量，使用权杖细胞计数

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面扩散和酵母染色体免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.