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. Sincronía a gran escala - óptimo para el Western Blot, Microarray / RNA-Seq, y otros ensayos intensivos de materiales <ol><li> De una acción congelador o una placa, crecer un 5 ml O / N de la cultura NA1000 cepa por agitación a 28 ° C en medio PYE. </li><li> Inocular 0,5 ml de las células de la etapa 1 en 25 ml de M2G (Tablas 1-2) y agitar a 28 ° C hasta que el cultivo alcanza una OD 600 entre 0,5 y 0,6. </li><li> Se inoculan las células en 1 l de M2G y agitar a 28 ° C. </li><li> Una vez DO600 alcanza de 0,5 a 0,6, confirmar la presencia de swarmer células utilizando microscopía fase líquida montada. Punto 1 l de células sobre un portaobjetos de vidrio, cubierta con una hoja de la cubierta, y la imagen por microscopía de fase. Confirmar la presencia de células swarmer mediante la visualización de las células que nadan rápidamente en la población. </li><li> Girar las células durante 15 min a 7 kxg a 4 ° C en un rotor JA-10. </li><li> Descartar el sobrenadante y añadir 180 ml de frío M2 (Tablas 1-2) y cosa suavementeuspend todas las células utilizando una pipeta serológica. Deseche células poco granuladas; que son las células predivisional y acechado. </li><li> Añadir 60 ml de solución de sílice coloidal frío (asegúrese de mezclar la suspensión de sílice coloidal bien antes de añadir a las células) y mezclar bien la suspensión celular. </li><li> Verter la suspensión de células en ocho tubos de 30 ml y centrifugar durante 30 min a 6,4 kxg a 4 ° C en un rotor JA-20. NOTA: Hay que ver a dos bandas distintas; la banda swarmer es la banda más baja mientras acechadas células / predivisional están en la banda superior (Figura 1B). </li><li> Aspirar con cuidado la banda superior y retire el líquido a ~ 1 cm por encima de la banda swarmer (la banda inferior). </li><li> (Crítica) Con una pipeta Pasteur, retire con cuidado la banda swarmer y colocar en un tubo limpio. Para lavar la sílice coloidal, parte superior del tubo con frío M2 y centrifugado durante 10 minutos a 6,4 kxg a 4 ° C en un rotor JA-20. </li><li> Desechar cuidadosamente el sobrenadante y resuspender ellas células en 20 ml de frío M2 y centrifugado durante 10 minutos a 6,4 kxg a 4 ° C en un rotor JA-20. </li><li> Resuspender todas las pastillas en 30 ml de frío M2 y medir la OD 600 usando un espectrofotómetro y en blanco utilizando medio M2 frío. Guardar 1 l para la imagen de fase para comprobar swarmer células; 90-95% de las células debería ser enjambres. </li><li> Centrifugar las células durante 5 minutos a 6,4 kxg a 4 ° C en un rotor JA-20. Resuspender las células en 28 ° C M2G medio de modo que el A 600 es de ~ 0,3-0,4 y comenzar agitación a 28 ° C. NOTA: Los rendimientos típicos son entre 30 y 60 ml de swarmer cultivo celular de 1 litro de células no sincronizadas. </li><li> Comience a tomar los puntos de tiempo (la cultura de tipo salvaje tomará aproximadamente 135-140 minutos para dividir) 8,9. En cada punto de tiempo, medir el OD 600. Compruebe que el OD 600 después de la división es de aproximadamente 2 veces la DO inicial 600. </li><li> Para ensayos de transferencia o de expresión génica occidentales, retirar alícuotas de 1 ml de la culture en los tiempos deseados, de girar a velocidad máxima en una centrífuga de mesa durante 30 segundos, rápidamente decantar o aspirar el medio, y el flash congelar el sedimento de células en nitrógeno líquido. Guarde las células a -80 ° C hasta el análisis de aguas abajo. </li></ol> 2. Sincronía pequeña escala - Óptimo para Microscopía <ol><li> De una acción congelador o una placa, crecer un cultivo de 5 ml O / N agitación a 28 ° C en M2G. </li><li> Diluir en 15 ml de M2G (Tablas 1-2) y crecer hasta mediados de log (OD 600 = 0,5 a 0,6). </li><li> Giran a 6,4 kxg durante 10 min a 4 ° C en un rotor JA-20, y se vuelve a suspender en 1 ml M2 frías (Tablas 1-2) y transferir a un tubo de microcentrífuga de 2 ml. </li><li> Giran a 15 kxg durante 3 min en un tubo de microcentrífuga para sedimentar las células, aspirar el sobrenadante, el sedimento se puso en hielo, y resuspender en 900 l de frío M2. </li><li> Añadir 900 l de PVP frío recubierto de sílice coloidal y centrifugado durante 20 minutos a 15 kxg a 4 ° C en un microtubo rocentrifuge. </li><li> (Crítica) Aspirar o una pipeta de la parte superior de banda celular acechado / predivisional y recoger la parte inferior swarmer banda en un nuevo tubo de microcentrífuga. </li><li> Lavar las células dos veces en swarmer 1 ml de frío, mientras que M2 centrifugación a 15 kxg durante 3 min. </li><li> Antes de que el centrifugado final, mover las células en un tubo de ensayo de vidrio de 1 ml pre-enfriada y medir la OD 600 de las células en comparación con un blanco de M2. </li><li> Resuspender el precipitado celular final a 28 ° C M2G a una DO entre 0,3 a 0,4 y agitar células / rollo a 28 ° C. NOTA: Los rendimientos típicos son entre 2 y 4 ml de cultivo celular swarmer. </li><li> Para los experimentos de microscopía, a la deseada puntos temporales lugar 1 l de células sobre una almohadilla de agarosa M2G para la imagen. </li></ol>

<ol><li>McAdams, H. H., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((System-level+design+of+bacterial+cell+cycle+control%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">System-level design of bacterial cell cycle control.</a> FEBS Lett. 583, 3984-3991 (2009).</li>
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<li>Evinger, M., Agabian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Envelope-associated+nucleoid+from+Caulobacter+crescentus+stalked+and+swarmer+cells%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells.</a> J. Bacteriol. 132, 294-301 (1977).</li>
<li>Tsai, J. W., Alley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteolysis+of+the+Caulobacter+McpA+chemoreceptor+is+cell+cycle+regulated+by+a+ClpX-dependent+pathway%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway.</a> J. Bacteriol. 183, 5001-5007 (2001).</li>
<li>Marks, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetic+basis+of+laboratory+adaptation+in+Caulobacter+crescentus%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">The genetic basis of laboratory adaptation in Caulobacter crescentus.</a> J. Bacteriol. 192, 3678-3688 (2010).</li>
<li>Ely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+Caulobacter+crescentus%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Genetics of Caulobacter crescentus.</a> Methods Enzymol. 204, 372-384 (1991).</li>
<li>Williams, B., Bhat, N., Chien, P., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ClpXP+and+ClpAP+proteolytic+activity+on+divisome+substrates+is+differentially+regulated+following+the+Caulobacter+asymmetric+cell+division%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division.</a> Mol. Microbiol. 93, 853-866 (2014).</li>
<li>Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Negative+control+of+bacterial+DNA+replication+by+a+cell+cycle+regulatory+protein+that+binds+at+the+chromosome+origin%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 120-125 (1998).</li>
<li>Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genes+directly+controlled+by+CtrA%2C+a+master+regulator+of+the+Caulobacter+cell+cycle%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 4632-4637 (2002).</li>
<li>Quon, K. C., Marczynski, G. T., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+cycle+control+by+an+essential+bacterial+two-component+signal+transduction+protein%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cell cycle control by an essential bacterial two-component signal transduction protein.</a> Cell. 84, 83-93 (1996).</li>
<li>Ferullo, D. J., Cooper, D. L., Moore, H. R., Lovett, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+cycle+synchronization+of+Escherichia+coli+using+the+stringent+response%2C+with+fluorescence+labeling+assays+for+DNA+content+and+replication%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.</a> Methods. 48, 8-13 (2009).</li>
<li>Degnen, S. T., Newton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+replication+during+development+in+Caulobacter+crescentus%5BTitle%5D)+AND+%22J.+Mol.+Biol%22%5BJournal%5D)">Chromosome replication during development in Caulobacter crescentus.</a> J. Mol. Biol. 64, 671-680 (1972).</li>
<li>Bates, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Escherichia+coli+baby+cell+column%3A+a+novel+cell+synchronization+method+provides+new+insight+into+the+bacterial+cell+cycle%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle.</a> Mol. Microbiol. 57, 380-391 (2005).</li>
<li>Abel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bi-modal+distribution+of+the+second+messenger+c-di-GMP+controls+cell+fate+and+asymmetry+during+the+caulobacter+cell+cycle%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Bi-modal distribution of the second messenger c-di-GMP controls cell fate and asymmetry during the caulobacter cell cycle.</a> PLoS Genet. 9, e1003744(2013).</li>
<li>Johnson, R. C., Ely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+spontaneously+derived+mutants+of+Caulobacter+crescentus%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Isolation of spontaneously derived mutants of Caulobacter crescentus.</a> Genetics. 86, 25-32 (1977).</li>
<li>Britos, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+response+to+carbon+starvation+in+Caulobacter+crescentus%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Regulatory response to carbon starvation in Caulobacter crescentus.</a> PLoS One. 6, e18179(2011).</li>
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</ol>

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. 
<p class='jove_...

El ciclo celular bacteriana controla tanto la replicación del genoma y la división de las células hijas. Es importante destacar que, como la resistencia a los antibióticos es una amenaza creciente para la salud pública, el ciclo celular bacteriana presenta un objetivo sin explotar para el desarrollo de antibióticos. En la bacteria Caulobacter crescentus, cada ciclo celular conduce a una división asimétrica, produciendo dos células hijas de diferentes destinos (Figura 1A) 1,2. Una célula hija hereda un flagelo y es móvil, mientras que la otra hija hereda un tallo y es sésiles. Un circuito genético integrado controla la progresión del ciclo celular y destino celular mediante la regulación transcripcional, fosfo-señalización, y proteólisis regulada 3. Además, la replicación de cromosomas y células hijas rendimiento segregación concurrentes que contienen exactamente una copia del cromosoma 4. Es importante destacar que estos dos tipos de células se pueden separar rápidamente por colloidal partícula de sílice centrifugación de densidad en la cepa NA1000 sincronizable 5-7 permitiendo el aislamiento de las células swarmer del resto de la población con altos rendimientos (Figura 1B). Aislado swarmer células luego proceder de forma sincrónica a través de la división celular asimétrica. Aquí, detallamos el protocolo utilizado para sincronizar Caulobacter cepa NA1000. Proporcionamos protocolos y sugerencias para solucionar problemas comunes para ambos de gran y pequeña escala sincronizaciones. Este procedimiento experimental proporciona una poderosa herramienta para interrogar el control espacio-temporal del ciclo celular Caulobacter y destino celular.

<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.

Sincronización típicamente produce dos bandas de células (Figura 1B): la banda swarmer, que tiene una densidad más alta, y una banda de células tallo / predivisional de menor densidad. Para asegurar los controles comunes de sincronización eficiente incluyen la supervisión de la OD 600 y la medición de los niveles de proteína CtrA por Western blot en momentos distintos del ciclo celular. La OD 600 debería aumentar en aproximadamente 2 veces durante el curso del ciclo celul...

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.

Sincronización de<em&gt; Caulobacter crescentus</em&gt; Para la Investigación del Ciclo Celular Bacteriana

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 vistas.  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.

Estudio de la expresión genética regulada por el ciclo celular mediante dos protocolos complementarios de sincronización celular

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

Investigación

Genética

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.

Imágenes de células vivas de Bacillus subtilis Y Streptococcus pneumoniae Automatizada utilizando Time-lapse microscopía

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

Inmunología e Infección

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.

Imágenes en vivo de la célula de fluorescencia de ciclo completo de células vegetativas de la lento-crecimiento Social bacteria 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 ...

Bioquímica

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.

Medición continua del ruido biológico en Escherichia Coli mediante microscopía de lapso de tiempo

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

Bioingeniería

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.

Microscopía de Fluorescencia de Células Vivas para Investigar la Localización de Proteínas Subcelulares y Cambios de Morfología Celular en Bacterias

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.

Seguimiento temporal de la progresión del ciclo celular El uso de citometría de flujo sin la necesidad de sincronización

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

Biología

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.

Vivo de la célula de microscopía de fluorescencia para observar procesos esenciales durante el crecimiento de la célula microbiana

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.

Combina la sincronización celular mitótica y microscopia Confocal de alta resolución para estudiar el papel de las proteínas multifuncional ciclo celular durante la 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 ...

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.

Sola célula microfluídicos análisis de 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.

Microirradiación láser para estudiar el reclutamiento de ADN durante la fase S

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 ...

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