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. Groß Synchrony - Optimal für Western Blot, Microarray / RNA-Seq und Sonstiges Material Intensive Tests <ol><li> Von einem Gefrierschrank Lager oder eine Platte, wachsen einem 5 ml O / N-Kultur des Stammes NA1000 durch Schütteln bei 28 ° C in PYE Medium. </li><li> Beimpfen von 0,5 ml der Zellen aus Schritt 1 in 25 ml M2G (Tabellen 1-2) und schüttelt bei 28 ° C, bis die Kultur eine OD 600 zwischen 0,5 und 0,6 erreicht. </li><li> Impfen Sie die Zellen in 1 l M2G und schütteln bei 28 ° C. </li><li> Sobald OD 600 erreicht 0,5 bis 0,6, bestätigen das Vorhandensein von Zellen mit Hilfe swarmer montiert flüssigen Phase Mikroskopie. Spot 1 ul der Zellen auf einen Objektträger, Deckel mit einem Deckglas und Bild durch Phasenmikroskopie. Bestätigen des Vorhandenseins von swarmer Zellen durch Visualisierung schneller schwimmen Zellen in der Population. </li><li> Spin-Zellen für 15 min bei 7 KXG bei 4 ° C in einem JA-10-Rotor. </li><li> Überstand verwerfen und fügen Sie 180 ml kaltes M2 (Tabellen 1-2) und sanft resuspend alle Zellen mit einer serologischen Pipette. Entsorgen Sie lose pelletierten Zellen; sie sind predivisional und stolzierte Zellen. </li><li> 60 ml kalte Kolloidales Siliciumdioxid-Lösung (unbedingt die Kolloidales Siliciumdioxid-Suspension auch vor der Zugabe zu den Zellen mischen) und mischen Sie die Zellsuspension gut. </li><li> Gießen Sie die Zellsuspension in acht 30-ml-Röhrchen und Spin für 30 min bei 6,4 KXG bei 4 ° C in einem JA-20 Rotor. HINWEIS: Man sollte zwei verschiedenen Bands zu sehen; die swarmer Band ist das untere Band während stolzierte / predivisional Zellen sind in der Top-Band (Abbildung 1B). </li><li> Die Top-Band vorsichtig absaugen und entfernen Sie die Flüssigkeit auf ca. 1 cm über dem swarmer Band (das untere Band). </li><li> (Kritisch) Mit einer Pasteur-Pipette, entfernen Sie vorsichtig die swarmer Band aufnehmen und in ein sauberes Röhrchen. Abzuwaschen Kolloidales Siliciumdioxid, oben das Rohr mit kaltem M2 und Schleudern für 10 min bei 6,4 KXG bei 4 ° C in einem JA-20-Rotor. </li><li> Vorsichtig den Überstand verwerfen und Resuspendieren derZellen in 20 ml kaltem M2 und Schleudern für 10 min bei 6,4 KXG bei 4 ° C in einem JA-20-Rotor. </li><li> Resuspendieren alle Pellets in 30 ml kaltem M2 und messen Sie die OD 600 mit einem Spektralphotometer und leere mit kaltem M2 Medium. Sparen Sie 1 ul für Phase-Bildgebung für swarmer Zellen überprüfen; 90-95% der Zellen sollten Schwärmer sein. </li><li> Anzentrifugieren die Zellen für 5 min bei 6,4 KXG bei 4 ° C in einem JA-20-Rotor. Zellen in 28 ° C M2G Medium, so dass der A 600 ist ~ 0,3-0,4 und beginnen bei 28 ° C schüttelnd. HINWEIS: Typische Ausbeuten zwischen 30 und 60 ml swarmer Zellkultur von 1L unsynchronisierten Zellen. </li><li> Fangen Sie an, Zeitpunkte (Wildtyp-Kultur wird in etwa 135 bis 140 Minuten in Anspruch nehmen zu teilen) 8,9. Zu jedem Zeitpunkt, messen Sie die OD 600. Prüfen, ob die OD 600 nach der Teilung ist ungefähr 2X die Anfangs-OD 600. </li><li> Für Western-Blot oder Genexpressionsassays entfernen 1 ml Aliquots der culture zu den gewünschten Zeitpunkten, Spin-down bei max Geschwindigkeit in einer Tischzentrifuge für 30 Sekunden, schnell Dekantieren des Mediums, und Flash Einfrieren des Zellpellets in flüssigem Stickstoff. Bewahren Sie die Zellen bei -80 ° C bis Downstream-Analyse. </li></ol> 2. Kleines Synchrony - Optimal für die Mikroskopie <ol><li> Von einem Gefrierschrank Lager oder eine Platte, wachsen ein 5 ml O / N-Kultur unter Schütteln bei 28 ° C im M2G. </li><li> Verdünnen Sie in 15 ml M2G (Tabellen 1-2) und wachsen, bis mid-log (OD 600 = 0,5-0,6). </li><li> Spin 6,4 KXG für 10 min bei 4 ° C in einem JA-20-Rotor und Resuspension in 1 ml kaltem M2 (Tabellen 1-2) und in ein 2 ml-Mikrozentrifugenröhrchen. </li><li> Spin bei 15 KXG für 3 min in ein Mikrozentrifugenröhrchen Zellen zu pelletieren, absaugen Überstand, legen Sie die Tablette auf Eis, und resuspendieren in 900 ul kaltem M2. </li><li> Hinzufügen 900 ul kaltem PVP beschichtet kolloidales Siliciumdioxid und Spin für 20 min bei 15 KXG bei 4 ° C in einem Mikrofonrocentrifuge Rohr. </li><li> (Critical) Verwerfen Sie pipettieren aus der Spitze marschierte / predivisional Zellband und sammeln Sie die unten swarmer Band in ein neues Mikrozentrifugenröhrchen. </li><li> Waschen der swarmer Zellen zwei Mal in 1 ml kaltem M2 während der Zentrifugation bei 15 KXG für 3 min. </li><li> Vor dem letzten Spin, verschieben Sie die Zellen in eine vorgekühlte 1 ml Reagenzglas und messen Sie die OD 600 der Zellen im Vergleich zu einem Rohling aus M2. </li><li> Resuspendieren die endgültige Zellpellet in 28 ° C M2G bei einer OD von 0,3 bis 0,4 und schütteln / Roll-Zellen bei 28 ° C. Anmerkung: Typische Ausbeuten liegen zwischen 2 und 4 ml swarmer Zellkultur. </li><li> Für die Mikroskopie Experimente, zum gewünschten Zeitpunkt Punkten Platz 1 ul der Zellen auf eine M2G Agarose-Pad für die Bildgebung. </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.

Die bakterielle Zellzyklus steuert sowohl die Replikation des Genoms und die Teilung der Tochterzellen. Wichtig ist, wie Antibiotika-Resistenz ist eine wachsende Bedrohung der öffentlichen Gesundheit stellt der bakteriellen Zellzyklus eine ungenutzte Ziel für Antibiotika-Entwicklung. In dem Bakterium Caulobacter crescentus, führt jede Zellzyklus in einer asymmetrischen Teilung, wodurch zwei Tochterzellen unterschiedlicher Schicksal (1A) 1,2. Eine Tochterzelle erbt eine Geißel und ist beweglich, während die andere Tochter erbt einen Stiel und sitzend. Eine integrierte genetische Schaltung steuert Zellzyklus und Zellschicksal von Transkriptionsregulation, Phospho-Signalisierung und regulierte Proteolyse 3. Zusätzlich Chromosomenreplikation und gleichzeitige Trennung Ausbeute Tochterzellen, die genau eine Kopie des Chromosoms 4 enthalten. Wichtig ist, dass diese beiden Zelltypen schnell durch collo getrennt werdenidal Silicateilchen Dichtezentrifugation im synchronisierbar NA1000 Stamm 5-7 ermöglicht die Isolierung der swarmer Zellen aus dem Rest der Population mit hohen Ausbeuten (1B). Isoliert swarmer Zellen dann synchron gehen durch asymmetrische Zellteilung. Hier haben wir ausführlich das Protokoll für die Synchronisierung von Caulobacter Belastung NA1000. Wir bieten Protokolle und gemeinsame Tipps zur Fehlerbehebung für Klein- und angelegte Synchronisationen. Das experimentelle Verfahren bietet ein leistungsfähiges Werkzeug, um die räumlich-zeitliche Kontrolle der Caulobacter Zellzyklus und Zellschicksal zu verhören.

<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.

Synchronisations ergibt typischerweise zwei Banden von Zellen (1B): der swarmer Band, das eine höhere Dichte hat, und eine gestielte / predivisional Zellenband mit geringerer Dichte. Um sicherzustellen, dass effiziente Synchronisation gemeinsame Kontrollen umfassen die Überwachung der OD 600 und Messung der Konzentrationen von Ctra Protein durch Western-Blot auf verschiedene Zellzykluszeitpunkten. Die OD 600 sollte um etwa 2-fach im Verlauf des Zellzyklus (Figur 2) zu erhöhen. Die Western-Blot für die Zellzyklushauptregulator Ctra ist eine sinnvolle Kontrolle, um eine gute Synchronität (Abbildung 2) zu überprüfen. Ctra im swarmer Zelle verwendet, um die DNA-Replikation zu blockieren und ist beim Einsetzen der DNA-Replikation 10 abgebaut. Ctra wird dann später in den Zellzyklus synthetisiert und aktiviert die Transkription einer Reihe von Entwicklungsgenen, einschließlich vieler Komponenten des Flagellums 11,12. Eine erfolgreiche Synchronisation wird tseine oszillierende Muster der Ctra Protein-Ebene. <img alt="Abbildung 1" src="/files/ftp_upload/52633/52633fig1.jpg" /> Abbildung 1. Der Zellzyklus von Caulobacter crescentus. (A) Eine Karikatur Schema des swarmer Zellzyklus. Die swarmer Zellen differenzieren sich in replikationskompetentem stolzierte Zellen durch Zurückziehen Pili, Auswerfen Geißeln und Initiierung der DNA-Replikation. Die Kreise und Theta im Inneren der Zelle Umrissen gezeigten Strukturen darstellen Ruhe und Replikation von Chromosomen. Zellen dann durch den Zellzyklus ein einheitliches, Geißel an der Stange gegenüber dem Stiel Fortschritt. Bei der Division, zwei einzigartige Zelltypen erzeugt werden, blockiert ein Replikations beweglichen swarmer Zelle und einer replikationskompetentem stationären stolzierte Zelle. (B) Repräsentative Ergebnisse für Dichtezentrifugation. Geringere Dichte gestielt und Predivisional Zellen schwimmen in der Nähe der Spitze des Gradienten, während dichte swarmer Zellen am Ende in Richtung der Unterseite des Rohres. Maßstabsbalken sind 1 &amp; mgr; m in der Phase-Mikroskopie-Aufnahmen. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> <img alt="Abbildung 2" src="/files/ftp_upload/52633/52633fig2.jpg" /> Abbildung 2. Repräsentative Ergebnisse für eine erfolgreiche swarmer Zelle synchron. Die Zellmasse durch OD 600 gemessen, sollte langsam zunehmen und schließlich im Verlauf des Assays zu verdoppeln. 1 ml Aliquots wurden in Kunststoffküvetten aus einer großen Zelle Synchronität und die OD 600 gemessen mit einem Spektrophotometer bei den angegebenen Zeitpunkten pipettiert. Zusätzlich sollte das Ctra Zellzyklusmaster regulatorisches Protein in der swarmer Zelle vorhanden sein, um die DNA-Replikation, gefolgt von einem schnellen Abbau blockierenübereinstimmend mit der Initiation der DNA-Replikation. Ctra wird dann später in den Zellzyklus regeneriert werden, um die Expression vieler Entwicklungsgenen, einschließlich kritischer Flagella und Pili Komponenten zu aktivieren. Western-Blots für die Zellzyklus-Regulatorprotein Master Ctra wurden hergestellt, indem 1 ml Aliquots einer großen Gleichtakt durchgeführt. Die Zellen wurden in 250 ul Probenpuffer Laemmlli pro OD 600 resuspendiert, auf einem 10% Tris-Gly-PAGE, auf PVDF übertragen getrennt und mit Anti-Ctra Antikörper geblottet. Fehlgeschlagen Synchronität Verfahren führen zu Ctra Western Blots ohne Veränderung der Protein-Ebene. α-Ctra Antikörper 12 wurde bei einer 1: 10.000-Verdünnung für 1,5 h in 3% Milch TBST und 3-mal gewaschen, in TBST. Goat-α-Kaninchen-Sekundär wurde dann bei 1 hinzugefügt: 10.000 Verdünnung in 3% Milch TBST 1 Stunde, 3 mal mit TBST gewaschen und auf Film aufgenommen mit einer Chemilumineszenznachweis Kit. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">Bitte</a>Klicken Sie hier, um eine größere Version dieser Figur zu sehen. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Na 2 HPO 4 </td><td> 17,4 g </td></tr><tr><td> KH 2 PO 4 </td><td> 10,6 g </td></tr><tr><td> NH 4 Cl </td><td> 10 g </td></tr><tr><td> H 2 O </td><td> Resuspendieren in 1 L &amp; Autoklav </td></tr></tbody></table> Tabelle 1 20X M2 Salze Rezept. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 20X M2 Salze </td><td> 50 ml </td><td></td></tr><tr><td> 0,5 M MgSO 4 </td><td> 1 ml </td><td></td></tr><tr><td> 20% Glucose </td><td> 10 ml </td><td> Ersatz für H 2 O in M2 </td></tr><tr><td> Eisensulfat Chelatlösung </td><td> 1 ml </td><td></td></tr><tr><td> 0,1 M CaCl &amp; sub2; </td><td> 5 ml </td><td> In letzter avoid Niederschlag </td></tr><tr><td> H 2 O </td><td> Füllen Sie bis zu 1 L </td><td></td></tr><tr><td> Sterilfilter mit 0,22 &amp; mgr; </td><td></td><td></td></tr></tbody></table> Tabelle 2. M2 und M2G Rezept.

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.

Synchronisation<em&gt; Caulobacter crescentus</em&gt; Für die Untersuchung der Bakterienzellzyklus

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 Aufrufe.  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.

Serie: 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.

Das Studium der Zellzyklus-regulierten Genexpression durch zwei komplementäre Zellsynchronisationsprotokolle

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

Forschung

Genetik

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.

Live Cell Imaging von Bacillus subtilis Und Streptococcus pneumoniae Verwendung von Automated Zeitraffer-Mikroskopie

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

Immunologie und Infektion

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.

Fluoreszenz Live Cell Imaging der komplette Vegetative Zellzyklus von langsam wachsenden sozialen Bakterium 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 ...

Biochemie

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.

Kontinuierliche Messung des biologischen Rauschens in Escherichia Coli mittels Zeitraffermikroskopie

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

Biotechnik

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.

Live-Cell Fluoreszenzmikroskopie zur Untersuchung subzellulärer Proteinlokalisierung und Zellmorphologie Veränderungen in Bakterien

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.

Zeitliche Verfolgung von Zellzyklus mittels Durchflusszytometrie, ohne die Notwendigkeit für die Synchronisation

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

Biologie

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.

Live Cell Fluoreszenzmikroskopie um wesentliche Vorgänge während der mikrobiellen Zellwachstum beobachten

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.

Kombination von mitotische Zelle Synchronisation und hochauflösenden konfokalen Mikroskopie zur Untersuchung der Rolle von multifunktionalen Zelle Zyklus Proteine während der Mitose

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.

Einzellige mikrofluidischen Analyse von Bacillus subtilis

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.

Laser-Mikrobestrahlung zur Untersuchung der DNA-Rekrutierung während der 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 ...

Synchronisation

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