Name: synchronization of caulobacter crescentus for investigation of the bacterial cell cycle
Uploaded: 2015-04-08T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.

1.大型同步性 - 最佳西方印迹，芯片/ RNA测序，和其他物资集约化化验<ol><li>从冰柜股票或板块，通过在28°C在PYE中颤抖成长应变NA1000的5毫升O / N培养。 </li><li>接种0.5ml所细胞从步骤1在25毫升M2G的（表1-2）和摇动在28℃，直到培养物达到0.5和0.6之间的OD 600。 </li><li>接种细胞到1升M2G和动摇，在28°C。 </li><li>一旦OD 600达到0.5至0.6，确认swarmer使用液相安装相位显微镜细胞的存在。点1微升的细胞在载玻片上的，盖上盖玻片，并通过图像相显微镜。通过在人口可视迅速游动细胞确认swarmer细胞的存在。 </li><li>在7 KXG在JA-10转子旋转细胞进行15分钟，在4℃。 </li><li>弃去上清液，加入冷M2的180毫升（表1-2），轻轻水库uspend所有使用血清吸管的细胞。放弃松散沉淀的细胞;他们是predivisional大步细胞。 </li><li>增加60毫升冷的胶体二氧化硅溶液（前务必同时增加了细胞混合胶体二氧化硅悬浮液）和混合细胞悬液好。 </li><li>倾细胞悬液分为八个30毫升管和在JA-20转子旋转30分钟，在6.4 KXG在4℃。 注:每个人都应该看到两个不同的频段;所述swarmer频带是低频带，而柄/ predivisional细胞是在顶带（ 图1B）。 </li><li>小心吸顶带，并取出液体约1厘米以上的swarmer频段（低频带）。 </li><li> （严重）用巴斯德吸管，小心地取出swarmer带，并放入一个干净的试管。洗去胶体二氧化硅，顶管断用冷M2和自旋10分钟，在6.4 KXG在4℃下在JA-20转子。 </li><li>小心弃去上清液，重悬细胞在20毫升冷M2的和自旋为在6.4 KXG在JA-20转子10分钟，在4℃。 </li><li>重悬的所有粒料于30毫升冷的M2的和使用分光光度计测量的OD 600和空白使用冷M2培养基。保存1微升相成像检查swarmer细胞;细胞的90-95％应是游动。 </li><li>在JA-20转子降速细胞5分钟，在6.4 KXG在4℃。悬浮细胞在28°C M2G中，这样的A 600为0.3-0.4〜并开始晃动，在28°C。 注:典型的产量是30和60毫升swarmer细胞培养，从非同步的细胞1L之间。 </li><li>开始服用的时间点（野生型文化将需要大约135-140分钟分）8,9。在每个时间点，测量OD 600。检查分割后的OD 600大约是2倍的初始OD 600。 </li><li>对于免疫印迹或基因表达分析，除去铜1毫升等分lture在所需的时间点，在台式离心机降速在最大速度为30秒，迅速倒出或者吸出培养基，并快速冷冻在液氮中的细胞沉淀。存储单元在-80°C，直到下游分析。 </li></ol> 2.小​​规模的同步性 - 最佳显微镜<ol><li>从冰柜股票或板块，成长5ml的O / N培养摇晃在28℃，M2G。 </li><li>稀释在15毫升M2G的（表1-2）和生长，直到数中期（OD 600 = 0.5-0.6）。 </li><li>旋在6.4 KXG为10分钟，在4℃下，在JA-20转子，和重悬在1ml冷的M2（表1-2），并转移到2ml微量离心管中。 </li><li>旋转15 KXG在离心管沉淀细胞，吸出上清液3分钟，放小球上的冰和重悬在900微升冷M2的。 </li><li>添加900微升冷的PVP在15 KXG涂覆的胶体二氧化硅和自旋20分钟，在4℃下在麦克风rocentrifuge管。 </li><li> （严重）吸或吸管从顶部大步/ predivisional细胞乐队和收集swarmer波段底部到一个新的离心管。 </li><li>在1ml冷的M2在15 KXG而离心3分钟洗swarmer细胞两次。 </li><li>在最后的旋转前，将细胞注入一预冷1毫升玻璃试管并测量细胞的OD 600相比M2的空白。 </li><li>重悬最终的细胞沉淀到28°C M2G 0.3之间的OD - 0.4摇/滚细胞在28°C。 注意:典型的产率是毫升swarmer细胞培养物2和4之间。 </li><li>显微镜的实验，在所希望的时间点处1微升的细胞到一个M2G琼脂糖垫用于成像。 </li></ol>

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The authors have nothing to disclose.

The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle. 
The protocol is quick and yields healthy synchronized cells. Other synchronization methods require long starvation of cells and rely on the stringent response to arrest the cell cycle in the same state, leading to cells that are dissimilar to cultures in logarithmic growth 13. While &#8220;baby machines&#8221; and other surface attachment methods have been used successfully14,15, they typically have low yields of synchronized cells. The density centrifugation protocols presented here allow higher purity and yields of synchronized C. crescentus NA1000 swarmer cells.
Additional complications can lead to potential problems throughout the synchrony procedure. Have a liquid mount phase microscope handy to optically check the purity of cells before synchronization and to check the purity of swarmers. A good synchrony will yield >95% swarmer cells. Check the OD600 on a spectrophotometer to ensure the cells are doubling during the time course of the synchrony. 
Cells don&#8217;t synchronize as well in the presence of antibiotics or some mutations. If the strain does not carry a replicating plasmid, try to remove the antibiotics from the growth medium to improve swarmer cell yield. Cells grown in the presence of antibiotics or mutant strains will also have a slower cell cycle, so it is important to check the division time by microscopy for comparison to healthy strains. 
Avoid vortexing the cells, as this will shear off the flagella and lower the quality of the synchrony. In preparation for the synchrony protocol don&#8217;t grow the cells above OD600 0.5. This ensures healthy growth that isn&#8217;t altered by high cell density. Be careful when aspirating/pipetting off supernatants containing the stalked and predivisional cell populations. In particular, do not to touch the swarmer band which may lead to contamination and/or a loss in yield of the swarmer cells. The cell pellet will be very loose in the first wash of cells after removing them from the Colloidal silica. Be careful when removing the supernatant not to disrupt the pellet.
Occasionally NA1000 cells can lose synchronization capacity due to a loss of DNA in the prophage region of the genome 7. In this case, no swarmer band is observed or the swarmer band intensity is dramatically reduced. If possible, collect cells if present in the swarmer band and streak onto a PYE plate to re-isolate the synchronizable cells. If working with a mutant strain, it is often useful to reintroduce the mutation into a fresh batch of NA1000. Conversely, mutation of critical cell cycle regulatory proteins can, in some cases, disrupt the ability of cells to synchronize16.
M2G medium was initially described with a higher 20 mM phosphate concentration 8,17; however, current studies use a lower 10 mM concentration as presented in Table 1 18,19. Alternative to the standard M2G medium, it is possible to use richer PYE medium. Here the unsynchronized growth and growth after synchronization can be substituted for PYE.
C. crescentus NA1000 provides a valuable experimental tool to study the bacterial cell cycle. Additionally, due to the single copy of the chromosome in the swarmer cell, this bacterium has also become a powerful model to study the structure of the bacterial chromosome.

细菌细胞周期控制的基因组中的两个复制和子细胞的分裂。重要的是，抗生素耐药性是一个日益严重的威胁到公众健康，细菌细胞周期呈现抗生素发展的尚未开发的目标。 在细菌柄杆菌新月 ，每个细胞周期导致一个不对称分裂，产生不同的命运（ 图1A）1,2的两个子细胞。一个子细胞继承了鞭毛是能动的，而另一个女儿继承柄和无柄是。一个集成的基因电路控制细胞周期进程和细胞命运的转录调控，磷酸化信号，和监管蛋白水解3。此外，染色体复制和并发分流率的子细胞包含4号染色体只有一个拷贝。重要的是，这两种类型的细胞可迅速地通过胶体分离在可同步NA1000菌株5-7允许swarmer细胞从具有高产率（ 图1B）的人口的其余部分隔离IDAL二氧化硅粒子密度离心。隔离swarmer细胞，然后通过不对称细胞分裂进行同步。在这里，我们详细介绍了协议用 ​​于同步柄杆菌菌株NA1000。我们提供的协议，并为大规模和小规模的同步常见故障排除技巧。这个实验的过程提供了一个功能强大的工具来询问柄杆菌细胞周期和细胞命运的时空控制。

<table><tbody><tr><td>PVP Coated Colloidal Silica (Percoll)</td><td>Sigma-Aldrich</td><td>P4937</td><td></td></tr><tr><td>Colloidal Silica (Ludox AS-40)</td><td>Sigma-Aldrich</td><td>420840</td><td></td></tr><tr><td>JA10 Rotor</td><td>Beckman-Coulter</td><td>369687</td><td></td></tr><tr><td>JA20 Rotor</td><td>Beckman-Coulter</td><td>334831</td><td></td></tr><tr><td>Ferrous Sulfate Chelate Solution</td><td>Sigma-Aldrich</td><td>F0518</td><td></td></tr><tr><td>30 ml Centrifuge Tubes</td><td>Corning</td><td>8445</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>SX0720-1</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>BDH9268-500G</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Amresco</td><td>0621-500g</td><td></td></tr></tbody></table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

同步通常产生细胞的两条带（ 图1B）:对swarmer带，它具有较高的密度和较低的密度的一个柄/ predivisional细胞带。为了确保高效的同步常见的控制包括监测OD 600和测量CTRA蛋白水平通过免疫印迹在不同的细胞周期的时间点。的OD 600应在细胞周期的过程中（ 图2）中约2倍的增加。在免疫印迹的细胞周期主控调节CTRA是一个有用的控制，以验证一个好的同步（ 图2）。 CTRA是利用在swarmer细胞阻断DNA复制的，对DNA复制10的发作被降解。 CTRA然后在细胞周期的后期合成并激活一个主机发育基因包括鞭毛11,12的许多组件的转录。一个成功的同步将有吨他的摆动CTRA蛋白水平的图案。 <img alt="图1" src="/files/ftp_upload/52633/52633fig1.jpg" /> 图1. 新月柄杆菌的细胞周期。（A）的swarmer细胞周期的卡通图。该swarmer细胞分化成复制能力大步细胞收回菌毛，鞭毛弹出，并启动DNA的复制。圆和细胞内的轮廓显示THETA结构代表了静态和复制染色体。细胞然后通过细胞周期建立在柄相对的磁极单鞭毛进步。在除法中，产生了两个独特的细胞类型，复制阻断能动swarmer细胞和具有复制能力的固定的柄细胞。（B），用于密度离心的代表性结果。下密度柄和predivisional细胞附近的梯度的顶部浮起而致密swarmer细胞最终朝向管的底部。比例尺为1微米阶段显微镜图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52633/52633fig2.jpg" /> 图对于成功swarmer小区同步2.代表性结果。通过OD 600测量的细胞块应缓慢增加，并最终增加一倍整个实验过程中。 1ml等分从大型小区同步，并使用分光光度计在指定的时间点测量的OD 600吸移到塑料比色皿。此外，CTRA细胞周期的主调控蛋白应存在于swarmer细胞阻断DNA复制，随后迅速降解重合DNA复制的起始。 CTRA然后在细胞周期以后再生来激活的许多发育基因的表达，包括临界鞭毛和菌毛组件。 Western印迹对细胞周期的主调控蛋白CTRA通过采取大规模同步的1ml等份进行。将细胞在250微升重新悬浮Laemmlli样品缓冲液每OD 600，分离在10％的TRIS-GLY PAGE，转移到PVDF，并印迹用抗CTRA抗体。未能同步程序导致CTRA印迹在蛋白水​​平没有变化。 α-CTRA抗体12温育以1:10,000稀释1.5小时中3％的牛奶的TBST和在TBST中洗涤3次。山羊-α的兔二次，然后在1中添加1:10,000稀释在3％的牛奶的TBST 1小时，洗涤3次用TBST，并用化学发光检测试剂盒上成像膜。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>的Na 2 HPO 4 </td><td> 17.4克</td></tr><tr><td> KH 2 PO 4 </td><td> 10.6克</td></tr><tr><td> 氯化铵 </td><td> 10克</td></tr><tr><td> H 2Ø </td><td>重悬在1升高压釜＆ </td></tr></tbody></table> 表1. 20X M2盐配方。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 20X M2盐</td><td> 50毫升</td><td></td></tr><tr><td> 0.5M的硫酸镁 </td><td> 1毫升</td><td></td></tr><tr><td> 20％葡萄糖</td><td> 10毫升</td><td>在M2的替代品与H 2 O </td></tr><tr><td>硫酸亚铁螯合物溶液</td><td> 1毫升</td><td></td></tr><tr><td> 0.1M的氯化钙 </td><td> 5毫升</td><td>最后添加至AVOID沉淀</td></tr><tr><td> H 2Ø </td><td>填充到1升</td><td></td></tr><tr><td>无菌过滤器以0.22微米的过滤器</td><td></td><td></td></tr></tbody></table> 表2. M2和M2G配方。

Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

synchronization-caulobacter-crescentus-for-investigation-bacterial

Universidad San Sebastian

Research

JoVE Journal

Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K 次观看.  Stanford University School of Medicine. Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

视频: Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

科研

遗传学

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

免疫与感染

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

生物化学

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

生物工程

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

生物学

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 ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

激光微辐射研究S阶段DNA招募

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

同步

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此视频中的章节

通过两个互补细胞同步方案研究细胞周期调控的基因表达

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

使用延时显微镜连续测量大肠杆菌中的生物噪声

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

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

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

单细胞微流控芯片分析枯草芽孢杆菌

激光微辐射研究S阶段DNA招募

同步

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通过两个互补细胞同步方案研究细胞周期调控的基因表达

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

活细胞荧光显微镜研究细菌的亚细胞蛋白定位和细胞形态变化

细胞周期进程的时间跟踪使用流式细胞仪，无需同步

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

有丝分裂细胞同步与高分辨共聚焦显微镜联合应用研究细胞周期蛋白在有丝分裂过程中的作用

单细胞微流控芯片分析枯草芽孢杆菌

激光微辐射研究S阶段DNA招募

使用延时显微镜连续测量大肠杆菌中的生物噪声