Name: synchronization of caulobacter crescentus for investigation of the bacterial cell cycle
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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. Büyük ölçekli Synchrony - Western Blot, Mikroarray / RNA-Sek, ve Diğer Malzeme Yoğun Tahliller için optimal <ol><li> Bir dondurucu stok veya bir plaktan, pye ortamı içinde 28 ° C&#39;de çalkalama ile streyn NA1000 5 ml&#39;lik bir O / N kültürünün yetiştirilmesi. </li><li> M2g 25 ml (Tablo 1-2) &#39;de, adım 1 hücreleri, 0.5 ml inoküle ve Kültür 0.5 ve 0.6 arasında bir OD 600 ulaşıncaya kadar 28 ° C&#39; de çalkalanır. </li><li> M2g 1 L hücreleri ile inoküle ve 28 ° C de çalkalanır. </li><li> OD 600 0,5-0,6 ulaştığında, sıvı monte faz mikroskobu kullanarak hücrelerin swarmer varlığını doğrulamaktadır. Faz mikroskobu ile bir cam slayt üzerinde hücrelerin 1 ul, bir kapak kayma ile kapak Noktası, ve görüntü. Nüfusun hızla yüzme hücrelerin görselleştirerek swarmer hücrelerinin varlığını doğrulamak. </li><li> JA-10 rotor içinde 4 ° C&#39;de 7 KXG 15 dakika boyunca hücreler dönerler. </li><li> Süpernatantı atın ve soğuk M2 180 ml ekleyin (Tablo 1-2) ve yavaşça resserolojik bir pipet kullanılarak tüm hücreleri uspend. Gevşek pelet hücreleri atın; bunlar predivisional ve saplı hücrelerdir. </li><li> Soğuk Kolloidal silika çözeltisi 60 ml ekleyin ve iyice karıştırın hücre süspansiyonu (iyi hücrelere eklemeden önce Kolloidal silika süspansiyonu karıştırmak için emin olun). </li><li> Sekiz 30 ml tüpler içinde hücre süspansiyonu dökün ve bir JA-20 rotor içinde 4 ° C&#39;de 6.4 KXG C&#39;de 30 dakika boyunca dönerler. NOT: Bir iki ayrı bantları görmelisiniz; saplı / predivisional hücreler üst bant (Şekil 1B) iken swarmer bant alt grubudur. </li><li> Dikkatle kapalı üst bandı aspire ve swarmer bant (alt bant) Yukarıda ~ 1 cm sıvıyı çıkarmak. </li><li> Pasteur pipeti kullanarak (Kritik), dikkatlice swarmer bandı çıkarın ve temiz bir tüpe yerleştirin. JA-20 rotor içinde 4 ° C&#39;de 6.4 KXG 10 dakika boyunca koloidal silis, iyi soğuk M2 ile tüp kapalı ve spin aşındırmak için. </li><li> Dikkatle süpernatant atmak ve tekrar süspansiyon20 soğuk M2 ml JA-20 rotor içinde 4 ° C&#39;de 6.4 KXG 10 dakika boyunca bir dönüş hücreler. </li><li> Soğuk M2 30 ml içine tüm pelet yeniden süspanse ve bir spektrofotometre kullanılarak OD 600 ölçmek ve soğuk M2 ortamı kullanarak boş. Hücreler swarmer kontrol etmek için faz görüntüleme için 1 ul kaydedin; Hücrelerin% 90-95 diğer canlılar olmalıdır. </li><li> JA-20 rotor içinde 4 ° C&#39;de 6.4 KXG 5 dakika boyunca hücreler dönerler. 28 ° C M2G ortam içinde yeniden süspanse edilen hücreler, 600 ~ 0.3-0.4 ve 28 ° C&#39;de çalkalanarak başlar, böylece. NOT: Tipik verimler 30 ve eşitlenmemiş hücrelerin 1L den swarmer hücre kültürü 60ml arasındadır. </li><li> Zaman noktaları (vahşi tip kültür bölmek için yaklaşık 135-140 dakika sürecektir) 8,9 alarak başlayın. Her zaman noktasında, OD 600 ölçer. Bölünmeden sonra OD 600 yaklaşık 2X başlangıç ​​OD 600 olduğunu kontrol edin. </li><li> Western blot ya da gen sentezleme tahlilleri için, cu 1 ml alikotları kaldırmaİstenen zaman noktalarında lture, hızlı bir şekilde 30 saniye için bir masa üstü santrifüjü içinde maksimum hızda aşağı spin süzün veya orta havalandırın, ve sıvı nitrojen içinde hücre pelletini dondurma. Alt baş analiz edilinceye kadar -80 ° C&#39;de hücreler saklayın. </li></ol> 2. Küçük ölçekli Synchrony - Mikroskopi için Optimal <ol><li> Bir dondurucu stok veya bir plaktan, 5 ml&#39;lik bir O / N kültür m2g 28 ° C&#39;de çalkalanarak büyür. </li><li> M2G 15 ml (Tablo 1-2) sulandırmak ve orta-log (OD = 0.5-0.6 600) kadar büyür. </li><li> 1 ml soğuk M2 (Tablo 1-2) ve 2 ml&#39;lik bir mikrosantrifüj tüpüne transfer JA-20 rotor içinde 4 ° C&#39;de 10 dakika boyunca 6.4 KXG dönerler ve süspansiyon haline getirin. </li><li> Süpernatantı aspire, hücreler topak haline getirilip, bir mikrosantrifüj tüpü içinde, 3 dakika için 15 KXG Spin soğuk M2 900 ul buz üzerinde pelet ve tekrar süspansiyon koydu. </li><li> Bir mikrofon 4 ° C&#39;de 15 KXG C&#39;de 20 dakika koloidal silis ve spin kaplama soğuk PVP 900 ul eklerocentrifuge tüp. </li><li> (Kritik) aspire garnitürü saplı / predivisional hücre bandı kapalı pipetlemeyin ve yeni bir mikrosantrifüj tüp içine bant swarmer alt toplamak veya. </li><li> 3 dakika boyunca 15 KXG santrifüj edilirken, M2, 1 ml swarmer hücreler iki kez yıkayın. </li><li> Son sıkma önce, önceden soğutulmuş bir 1 ml&#39;lik bir cam deney tüpü içine hücreleri taşımak ve M2 bir boşluk ile karşılaştırıldığında hücrelerin OD 600 ölçer. </li><li> 0,3 arasında bir OD 28 ° C M2G içine son hücre pelletini - 0.4 ve 28 ° C&#39;de / rulo hücreleri sallayın. Not: Tipik verimler swarmer hücre kültürünün 2 ila 4 ml dir. </li><li> Görüntüleme için bir M2G agaroz ped üzerine hücrelerin istenilen zaman noktaları yer 1 ul mikroskopi deneyleri için. </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.

bakteriyel hücre döngüsü genomunun çoğaltma ve kızı hücrelerinin bölünmesini kontrol eder. Antibiyotik direnci halk sağlığı için büyüyen bir tehdit olarak önemlisi, bakteri hücre döngüsü antibiyotik gelişimi için bir hedef kullanılmayan sunuyor. Bakteri Caulobacter crescentus, her bir hücre döngüsü Farklı kaderi (Şekil 1A) 1,2 iki kız hücre sağlar, asimetrik bölünmesi yol açar. Bir yavru hücre, bir kamçı miras ve diğer kızı sap miras ve sapsız ise hareketlidir. Entegre devre genetik transkripsiyon regülasyonu, fosfor-sinyalizasyon ve düzenlenmiş proteoliz 3 hücre döngüsü ilerlemesini ve hücre kaderi kontrol eder. Buna ek olarak, kromozom replikasyonu ve eşzamanlı ayrımcılık verim kızı hücreleri kromozom 4 tam bir kopyasını içeren. Önemli bir şekilde, bu iki hücre tipi hızla collo ile ayrılabiliryüksek verim (Şekil 1B) ile nüfusun geri kalanından swarmer hücrelerinin izolasyonu sağlayan synchronizable NA1000 suşu 5-7 idal silika parçacık yoğunluğu santrifüj. Hücrelerin swarmer İzole sonra asimetrik hücre bölünmesi yoluyla eşzamanlı devam edin. Burada, biz detay protokolü Caulobacter suşu NA1000 senkronize etmek için kullanılır. Biz protokolleri ve büyük ve küçük ölçekli her iki eşitleme için ortak sorun giderme ipuçları sağlar. Bu deneysel prosedür Caulobacter hücre döngüsü ve hücre kaderinin kontrolünü zamanmekansal sorgulamak için güçlü bir araç sağlar.

<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.

Daha yüksek bir yoğunluğa sahip swarmer bandı, ve daha düşük yoğunluklu bir saplı / predivisional hücre bandı: eşitleme genellikle hücrelerin iki bant (Şekil 1B) verir. Etkili bir senkronizasyon ortak kontrol OD 600 izleme ve farklı hücre döngüsü zaman noktalarında western blot ile CtrA&#39;nın protein seviyelerinin ölçülmesi dahildir sağlamak. OD 600, hücre döngüsü sırasında (Şekil 2) boyunca, yaklaşık 2 kat artış gerekir. hücre döngüsü ana regülatör CtrA&#39;nın için batı leke iyi senkronizasyonunu (Şekil 2) doğrulamak için yararlı bir kontroldür. CtrA&#39;nın DNA replikasyonunu bloke etme swarmer hücre kullanılmaktadır ve DNA replikasyonu 10 başlamasından sonra düşer. CtrA&#39;nın daha sonra hücre döngüsünün sentezlenmiş ve flagellum&#39;un 11,12 birçok bileşenleri de dahil olmak üzere gelişme genlerin bir ev sahibi transkripsiyonunu aktive eder. Başarılı bir senkronizasyonu t olacaktırCtrA&#39;nın protein seviyelerinin onun salınım düzeni. <img alt="Şekil 1," src="/files/ftp_upload/52633/52633fig1.jpg" /> Şekil 1. Caulobacter crescentus hücre döngüsü (A). Swarmer hücre döngüsünün bir çizgi şematik. swarmer hücreleri, pili geri çekilmesi flagella çıkarılması ve DNA replikasyonunu başlatan olarak replikasyon yapabilen bir saplı hücreleri içinde farklılık gösterir. çevreler ve hücre hatları içinde gösterilen teta yapılar sakin ve kopyalayan kromozom temsil eder. Hücreler daha sonra sap zıt kutbunda tek kamçılı bina, hücre döngüsü yoluyla ilerleme. Bölünme üzerine, iki eşsiz hücre tipleri üretilir, bir çoğaltma (B) yoğunluk santrifüj için Temsilcisi sonuçları.. Hücre ve bir çoğaltma yetkili sabit saplı hücrenin swarmer hareketli bloke Alt yoğunluk saplı ve predivhücrelerin swarmer yoğun tüpün altına doğru sonuna kadar ise isional hücreler degrade üstüne yakın yüzer. Ölçek çubukları faz mikroskobu görüntüleri 1 mikron bulunmaktadır. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52633/52633fig2.jpg" /> Şekil hücre senkronizasyonu swarmer başarılı 2. Temsilcisi sonuçları. OD 600 ile ölçülen hücre kütlesi yavaş yavaş artırmak ve sonuçta testinin boyunca iki katına çıkacaktır. 1 ml&#39;lik eş payları, bir büyük hücreli eşzamanlı ve belirtilen zaman noktalarında bir spektrofotometre kullanılarak ölçülen OD 600 plastik küvetler içine pipetlenmiştir. Buna ek olarak, CtrA&#39;nın hücre döngüsü ana düzenleyici protein, bir süratli bir degradasyonu ile, ardından DNA replikasyonunu bloke etmek swarmer hücresinde mevcut olmalıdırDNA replikasyonunun başlatılması ile çakışık. CtrA&#39;nın o zaman kritik kamçılı ve pili bileşenleri de dahil olmak üzere pek çok gelişme genin ifadesinin aktive edilmesi için, daha sonra hücre döngüsü yeniden üretilir. Hücre döngüsü ana düzenleyici protein CtrA Western blotlar büyük ölçekli senkronize 1 ml&#39;lik numuneler alınarak yapıldı. Hücreler PVDF transfer% 10 TRIS-GLY PAGE, üzerinde ayrıldı, OD 600 250 ul Laemmlli numune tamponu yeniden süspansiyon haline getirildi, ve anti-CtrA&#39;nın antikoru ile lekelenir. Başarısız senkron işlemleri protein düzeylerinde değişiklik CtrA&#39;nın Western lekeleme yol açar. α-CtrA&#39;nın antikor 12 bir 1 ° C enkübe edilmiştir:% 3 süt TBST içinde 1.5 saat için 10,000 seyreltme ve TBST içinde 3 kez yıkanmıştır. Keçi α-Tavşan daha sonra 1 ilave edildi ikincil:. 1 saat için 10,000 seyrelti,% 3 süt TBST, TBST ile 3 kez yıkandı ve kemilüminesan deteksiyon kiti kullanılarak film görüntüsü <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">, lütfen</a>Bu rakamın büyük halini görmek için buraya tıklayın. <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 gr </td></tr><tr><td> NH4CI </td><td> 10 g </td></tr><tr><td> H2O </td><td> 1 L &amp; otoklav içinde süspanse </td></tr></tbody></table> Tablo 1. 20X M2 Tuzları Tarif. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 20X M2 Tuzlar </td><td> 50 mi </td><td></td></tr><tr><td> 0.5 M MgSO 4 </td><td> 1 mi </td><td></td></tr><tr><td> % 20 Glukoz </td><td> 10 mi </td><td> M2, H2O için Yedek </td></tr><tr><td> Demir Sülfat Şelat Çözüm </td><td> 1 mi </td><td></td></tr><tr><td> 0.1 M CaCl2 </td><td> 5 mi </td><td> Av son ekleoid yağış </td></tr><tr><td> H2O </td><td> 1 L doldurun </td><td></td></tr><tr><td> 0.22 uM filtre ile steril filtre </td><td></td><td></td></tr></tbody></table> Tablo 2. M2 ve M2G tarifi.

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.

Senkronizasyon<em&gt; Caulobacter crescentus</em&gt; Bakteriyel Hücre Döngüsü Soruşturma

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 ...

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Universidad San Sebastian

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Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K Görüntüleme.  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.

Video: 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.

Hücre Çevrimiyle Düzenlenen Gen İfadesini İki Tamamlayıcı Hücre Senkronizasyon Protokolü İle Çalışmak

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

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.

Canlı Hücre Görüntüleme Bacillus subtilis Ve Streptococcus pneumoniae Otomatik Zaman atlamalı Mikroskopi

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

İmmünoloji ve Enfeksiyon

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.

Floresans Live-hücre görüntüleme, tam bitkisel hücre döngüsü yavaş büyüyen sosyal bakteri 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 ...

Biyokimya

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.

Zaman Atlamalı Mikroskopi Kullanılarak Escherichia Coli'de Biyolojik Gürültünün Sürekli Ölçümü

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

Biyomühendislik

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.

Bakterilerde Hücre Altı Protein Lokalizasyonu ve Hücre Morfolojisi Değişikliklerini Araştırmak Için Canlı Hücre Floresan Mikroskobu

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.

Senkronizasyon Need for olmadan Akım Sitometri Kullanımı Hücre Döngüsü ilerleme zamansal İzleme

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

Biyoloji

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.

Temel işlemler sırasında mikrobiyal hücre büyümesini gözlemlemek için hücre Floresans mikroskobu canlı

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.

Mitotik hücre eşitleme ve yüksek çözünürlüklü Confocal mikroskobu mitoz sırasında çok fonksiyonlu hücre döngüsü proteinlerin rolü çalışmaya birleştirme

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.

Bacillus subtilis tek hücreli mikrosıvısal analizi

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 Aşamasında DNA İşe Alımını İncelemek için Lazer Mikro Işınlama

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 ...

Senkronizasyon

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