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.大規模同期性 - ウエスタンブロット、マイクロアレイ/ RNA-SEQ、およびその他素材集中アッセイに最適<ol><li>冷凍庫ストック又はプレートから、PYE培地で28℃で振とうすることにより歪NA1000の5ミリリットルO / N培養を成長。 </li><li> M2Gの25ミリリットル（表1-2）に、ステップ1からの細胞の0.5ミリリットルを接種し、培養が0.5と0.6の間のOD 600に達するまで28℃で振る。 </li><li> M2Gの1リットル中に細胞を接種し、28℃で振る。 </li><li> OD 600が 0.5〜0.6に達すると、液体取り付けられた位相差顕微鏡を用いて細胞swarmerの存在を確認する。位相差顕微鏡によってスライドガラス上の細胞の1μlを、カバーガラスで覆い、スポット、および画像。人口が急速に泳いで細胞を視覚化することによってswarmer細胞の存在を確認してください。 </li><li> JA-10ローターで4℃で7 kxgで15分間細胞をスピン。 </li><li>上清を捨て、冷たいM2（表1-2）の180ミリリットルを加え、穏やかにresは血清学的ピペットを使用してすべてのセルをuspend。緩くペレット化した細胞を捨てる。彼らはpredivisionalとストーカー細胞である。 </li><li>冷たいコロイダルシリカ溶液の60ミリリットルを加え、よく細胞懸濁液を混ぜる（よく細胞に添加する前にコロイダルシリカ懸濁液を混在してください）​​。 </li><li> 8 30ミリリットルチューブに細胞懸濁液を注ぎ、JA-20ローターで、4℃で6.4 kxgで30分間スピン。 注：Oneは、2つの異なるバンドが表示されるはずです。ストーカー/ predivisional細胞は、トップバンド（ 図1B）にいる間swarmer帯は下のバンドである。 </li><li>慎重にトップバンドをオフに吸引し、swarmerバンド（下のバンド）上記〜1 cmの液体を除去する。 </li><li>パスツールピペットを用いて（クリティカル）、慎重にswarmerバンドを削除して、清潔なチューブに配置します。 JA-20ローターで、4℃で6.4 kxgで10分間コロイダルシリカ、トップ冷たいM2とチューブオフとスピンを洗い流す。 </li><li>注意深く上清を破棄し、再懸濁JA-20ローターで冷たいM2の20ミリリットルと、4℃で6.4 kxgで10分間スピンのセル。 </li><li>コールドM2 30mlの中に全てのペレットを再懸濁し、分光光度計を用いてOD 600を測定し、冷M2培地を用いてブランク。 swarmer細胞を確認するために位相イメージングのための1μLを保存します。細胞90〜95％がswarmersでなければなりません。 </li><li> JA-20ローターで、4℃で6.4 kxgで5分間、細胞をスピンダウン。 28℃M2G培地中の細胞を再懸濁A 600になるように、〜0.3〜0.4と28℃で振とうを開始。 注：典型的な収量は30と同期していない細胞の1Lからswarmer細胞培養の60mLの間にある。 </li><li> 8,9（野生型の文化が分裂するのに約135〜140分かかります）の時点を取って開始します。各時点で、OD 600を測定する。分割後のOD 600が約2倍初期OD 600であることを確認してください。 </li><li>ウェスタンブロットまたは遺伝子発現アッセイのために、立方1mLのアリコートを除去所望の時点でlture、30秒間、卓上遠心分離機において最大速度でスピンダウンし、急速に培地をデカントまたは吸引し、フラッシュは、液体窒素中で細胞ペレットを凍結する。下流の分析まで-80℃で細胞を保管してください。 </li></ol> 2.小​​規模同期性 - 顕微鏡用に最適<ol><li>冷凍庫ストック又はプレートから、M2Gに28℃で振とう5ミリリットルO / N培養を成長。 </li><li> M2G（表1-2）15mlに希釈し、中期対数（OD 600 = 0.5〜0.6）になるまで成長する。 </li><li> JA-20ローター中で4℃で10分間6.4 kxgでスピンし、1ミリリットル中に再懸濁冷たいM2（表1-2）と2 mlのマイクロ遠心チューブに移す。 </li><li>上清を吸引し、細胞をペレットにマイクロ遠心チューブ中で3分間15 kxgでのスピンは、冷たいM2の900μlの氷の上でペレット、再懸濁を置く。 </li><li>マイクで4℃で15 kxgで20分間、コロイダルシリカとスピンコーティングされた冷たいPVP900μlのを追加します。rocentrifugeチューブ。 </li><li> （クリティカル）を吸引したり、トップを切っピペット/ predivisional細胞バンドをストーカーして、新しいマイクロ遠心チューブにバンドswarmer底に集める。 </li><li> 3分間15 kxgで遠心分離しながら、冷たいM2の1ミリリットル中swarmer細胞を2回洗浄します。 </li><li>最後のスピンの前に、予備冷却した1mlのガラス試験管に細胞を移動し、M2のブランクと比較した細胞のOD 600を測定する。 </li><li> 0.4及び28℃/ロール細胞を振る - 0.3の間ODで28℃、M2Gに最終的な細胞ペレットを再懸濁。 注：典型的な収量はswarmer細胞培養の2および4 mlの間である。 </li><li>顕微鏡実験のために、所望の時間にポイントがイメージングのためM2Gアガロースパッド上に細胞を1μLを置く。 </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. 
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細菌の細胞周期は、ゲノムの複製と娘細胞の分裂の両方を制御する。抗生物質耐性は、公衆の健康に成長している脅威であるとして重要なのは、細菌の細胞周期は、抗生物質開発のための未開発の目標を提示します。 細菌カウロバクターcrescentusでは、各細胞周期の異なる運命（ 図1A）1,2の2つの娘細胞を生じる、非対称分裂に導く。 One娘細胞は鞭毛を継承し、他の娘は茎を継承し、固着されている間運動性である。集積回路の遺伝子は、転写調節、リン酸化シグナル伝達、およびタンパク質分解規制3ことによって、細胞周期の進行および細胞の運命を制御する。また、染色体複製と同時分離は、第4染色体の正確に一つのコピーを含む娘細胞が得られる。重要なことに、これらの2つの細胞型は急速COLLOによって分離することができる高収率（ 図1B）を有する集団の残りからswarmer細胞の単離を可能にする同期可能NA1000株5-7 idalシリカ粒子の密度遠心分離。細胞s​​warmer分離された後、非対称細胞分裂を通じて同期的に進む。ここでは、詳細プロトコルは、 カウロバクター株 NA1000を同期させるために使用される。私たちは、プロトコルおよび大規模および小規模の両方の同期のための一般的なトラブルシューティングのヒントを提供する。この実験手順は、 カウロバクター細胞周期と細胞の運命の時空間制御を調べるための強力なツールを提供します。

<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.

より高い密度を有するswarmerバンド、およびより低い密度のストーカー/ predivisionalセル帯域：同期は、典型的には、セルの二つのバンド（ 図1B）が得られる。効率的な同期を確実にするために共通のコントロールがOD 600を監視し、異なる細胞周期の時点でウエスタンブロットによりCTRAタンパク質のレベルを測定することを含む。 OD 600は、細胞周期（ 図2）</s...

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.

の同期<em&gt;カウロバクターCrescentus</em&gt;細菌細胞周期の調査のため

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

Research

JoVE Journal

Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K ビュー.  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.

ビデオ: 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.

2つの相補的細胞同期プロトコルによる細胞周期制御遺伝子発現の研究

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

遺伝学

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.

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

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

免疫・感染学

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.

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

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.

タイムラプス顕微鏡による 大腸菌 における生物学的ノイズの連続測定

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

生物工学

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.

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

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.

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

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.

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

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.

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

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.

枯草菌の単一細胞マイクロ流体解析

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.

S期におけるDNA採用を研究するレーザーマイクロ照射

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 ...

の同期

要約

さらに動画を探す

この動画の章

2つの相補的細胞同期プロトコルによる細胞周期制御遺伝子発現の研究

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

タイムラプス顕微鏡による大腸菌における生物学的ノイズの連続測定

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

枯草菌の単一細胞マイクロ流体解析

S期におけるDNA採用を研究するレーザーマイクロ照射

の同期

要約

さらに動画を探す

この動画の章

2つの相補的細胞同期プロトコルによる細胞周期制御遺伝子発現の研究

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

タイムラプス顕微鏡による 大腸菌 における生物学的ノイズの連続測定

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

同期を必要とせずに、フローサイトメトリーを用いた細胞周期の進行の時間追跡

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

枯草菌の単一細胞マイクロ流体解析

S期におけるDNA採用を研究するレーザーマイクロ照射

タイムラプス顕微鏡による大腸菌における生物学的ノイズの連続測定