Name: synchronization of caulobacter crescentus for investigation of the bacterial cell cycle
Uploaded: 2015-04-08T15:00:00.000+00:00
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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. בתיאום בקנה מידה גדולה - אופטימלי עבור מערבי כתם, Microarray / RNA-Seq, ומבחנים עתירי חומרים אחרים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממניות מקפיא או צלחת, לגדול 5 מיליליטר O / תרבות N של NA1000 מתח על ידי טלטול על 28 מעלות צלזיוס במדיום פאי. </li><li style=";text-align:right;direction:rtl"> לחסן 0.5 מיליליטר של התאים משלב 1 ב 25 מיליליטר של M2G (לוחות 1-2) ולנער בגיל 28 ° C עד התרבות מגיעה OD 600 בין 0.5 ו -0.6. </li><li style=";text-align:right;direction:rtl"> לחסן את התאים לתוך 1 ליטר של M2G ולנער על 28 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> ברגע שמגיע OD 600 0.5-0.6, לאשר את קיומו של swarmer תאים באמצעות מיקרוסקופיה שלב הנוזלית רכוב. מקום 1 μl של תאים בשקופית זכוכית, לכסות עם תלוש כיסוי, ותמונה על ידי מיקרוסקופ שלב. לאשר את קיומם של תאי swarmer על ידי הדמיה תאים במהירות השחייה באוכלוסייה. </li><li style=";text-align:right;direction:rtl"> ספין תאים במשך 15 דקות בשעה 7 kxg על 4 מעלות צלזיוס בJA-10 הרוטור. </li><li style=";text-align:right;direction:rtl"> בטל supernatant ולהוסיף 180 מיליליטר של M2 הקר (לוחות 1-2) ומיל בעדינותuspend כל התאים באמצעות pipet סרולוגיות. בטל תאי pelleted רופף; הם תאי predivisional וצעדה. </li><li style=";text-align:right;direction:rtl"> להוסיף 60 מיליליטר של תמיסת סיליקה Colloidal הקרה (הקפד לערבב את ההשעיה סיליקה Colloidal היטב לפני הוספה לתאים) ומערבבים את ההשעיה התא היטב. </li><li style=";text-align:right;direction:rtl"> יוצקים את ההשעיה התא לשמונה 30 מיליליטר צינורות וספין למשך 30 דקות ב 6.4 kxg על 4 מעלות צלזיוס ב20-JA הרוטור. הערה: יש לראות את שתי להקות שונות; להקת swarmer היא הרצועה התחתונה בזמן שתאים ארבו / predivisional נמצאים בלהקה העליונה (איור 1). </li><li style=";text-align:right;direction:rtl"> בזהירות לשאוב הלהקה העליונה והסר את הנוזל ל~ 1 סנטימטר מעל ללהקת swarmer (הלהקה התחתונה). </li><li style=";text-align:right;direction:rtl"> (קריטי) שימוש pipet פסטר, להסיר בזהירות את להקת swarmer ומקום לתוך צינור נקי. כדי לשטוף את סיליקה Colloidal, ראש הצינור את עם M2 הקר וספין במשך 10 דקות ב 6.4 kxg על 4 מעלות צלזיוס ב20-JA הרוטור. </li><li style=";text-align:right;direction:rtl"> להשליך בזהירות את supernatant ו resuspendתאים ב 20 מיליליטר של M2 הקר וספין במשך 10 דקות ב 6.4 kxg על 4 מעלות צלזיוס ב20-JA הרוטור. </li><li style=";text-align:right;direction:rtl"> Resuspend כל כדורים לתוך 30 מיליליטר של M2 הקר ולמדוד את OD 600 באמצעות ספקטרופוטומטר וריק באמצעות מדיום M2 קר. להציל 1 μl הדמיה שלב כדי לבדוק swarmer תאים; 90-95% מתאים צריכים להיות swarmers. </li><li style=";text-align:right;direction:rtl"> ספין למטה התאים 5 דקות בkxg 6.4 על 4 מעלות צלזיוס ב20-JA הרוטור. תאי Resuspend ב -28 ° C בינוני M2G כך ש600 הוא ~ 0.3-0.4 ולהתחיל רועד ב 28 מעלות צלזיוס. הערה: תשואות אופייניות הן בין 30 ל 60 מ&quot;ל של swarmer תרבית תאים מ1 הליטר של תאים לא מסונכרנים. </li><li style=";text-align:right;direction:rtl"> להתחיל לקחת נקודות זמן (תרבות מהסוג בר תיקח כ 135-140 דקות לחלק) 8,9. בכל נקודת זמן, למדוד את OD 600. בדוק שOD 600 לאחר החלוקה הוא כ 2X OD הראשוני 600. </li><li style=";text-align:right;direction:rtl"> עבור מבחני כתם או ביטוי גנים מערביים, להסיר aliquots 1mL של מ&quot;קlture בנקודות הזמן הרצוי, ספין למטה במהירות מקסימלית בצנטריפוגה שולחן למשך 30 שניות, מהירות למזוג או לשאוב את המדיום, ופלאש להקפיא את התא גלולה בחנקן נוזלי. אחסן את התאים ב -80 ° C עד ניתוח במורד הזרם. </li></ol> 2. בתיאום בקנה מידה קטנה - אופטימלי עבור מיקרוסקופית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ממניות מקפיא או צלחת, לגדול 5 מיליליטר O / תרבות N רועדת ב 28 ° C בM2G. </li><li style=";text-align:right;direction:rtl"> לדלל ב -15 מיליליטר של M2G (לוחות 1-2) ולגדול עד אמצע יומן (OD 600 = 0.5-0.6). </li><li style=";text-align:right;direction:rtl"> ספין 6.4 kxg במשך 10 דקות ב 4 ° C ב20-JA הרוטור, וגלול ב 1 מיליליטר M2 (לוחות 1-2) קרים ולהעביר צינור microcentrifuge 2 מיליליטר. </li><li style=";text-align:right;direction:rtl"> ספין ב 15 kxg במשך 3 דקות בצינור microcentrifuge לגלולת תאים, לשאוב את supernatant, לשים את הכדור על קרח, וגלול ב900 μl של M2 הקר. </li><li style=";text-align:right;direction:rtl"> להוסיף 900 ​​μl של PVP הקר מצופה סיליקה וספין colloidal במשך 20 דקות ב 15 kxg על 4 מעלות צלזיוס במיקרופוןצינור rocentrifuge. </li><li style=";text-align:right;direction:rtl"> (קריטי) לשאוב או pipet את החלק העליון ארבה / להקת תא predivisional ולאסוף התחתון swarmer להקה לתוך צינור microcentrifuge חדש. </li><li style=";text-align:right;direction:rtl"> שוטף את תאי swarmer שתי פעמים ב 1 מיליליטר של M2 הקר תוך צנטריפוגה ב 15 kxg במשך 3 דקות. </li><li style=";text-align:right;direction:rtl"> לפני הסיבוב האחרון, להעביר את התאים לתוך מבחנת זכוכית 1 מיליליטר מראש צונן ולמדוד את OD 600 של התאים בהשוואה לריק של M2. </li><li style=";text-align:right;direction:rtl"> Resuspend תא גלולה הסופי ל -28 מעלות צלזיוס M2G בOD בין 0.3-0.4 ולנער תאים / גליל ב 28 מעלות צלזיוס. הערה: תשואות אופייניות הן בין 2 ל 4 מיליליטר של תרבית תאי swarmer. </li><li style=";text-align:right;direction:rtl"> לניסויים במיקרוסקופ, בμl 1 מקום נקודות זמן הרצוי של תאים על גבי משטח agarose M2G להדמיה. </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.

מחזור תא החיידק שולט שני השכפול של הגנום וחלוקת תאי בת. חשוב לציין, כעמידות לאנטיביוטיקה היא איום הולך וגדל לבריאות ציבור, מחזור תא החיידק מציג יעד שלא נוצל לפיתוח אנטיביוטיקה. בcrescentus Caulobacter החיידק, כל מחזור תא מוביל לחלוקה סימטרית, מניב שני תאי בת של גורלות שונים (איור 1 א) 1,2. תא בת אחת יורש השוטון והוא ניע ואילו הבת אחרת יורשת גבעול ונייח. מעגל משולב גנטי שולט התקדמות מחזור התא ותא גורל על ידי רגולציה תעתיק, phospho-איתות, וproteolysis המוסדרת 3. בנוסף, שכפול כרומוזום ותאים בת תשואת הפרדה במקביל המכילים עותק אחד בדיוק של כרומוזום 4. חשוב לציין, אלה שני סוגי התאים ניתן להפריד במהירות על ידי Colloצנטריפוגה idal חלקיקי סיליקה צפיפות במתח NA1000 synchronizable 5-7 המאפשר בידוד של תאי swarmer משאר האוכלוסייה עם תשואות גבוהות (איור 1). מבודד swarmer תאים ולאחר מכן להמשיך באופן סינכרוני באמצעות חלוקת תא סימטרית. כאן, אנו פירוט הפרוטוקול המשמשים לסנכרון NA1000 מתח Caulobacter. אנו מספקים פרוטוקולים ועצות לפתרון בעיות משותפות לשני סנכרונים מידה גדול וקטנה בקנה מידה. הליך ניסיוני זה מספק כלי רב עוצמה כדי לחקור את שליטת spatiotemporal של מחזור תא Caulobacter וגורל תא.

<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.

סנכרון בדרך כלל תשואות שתי להקות של תאים (איור 1): להקת swarmer, שבו יש צפיפות גבוהה יותר, ולהקה ארבה / predivisional תא של צפיפות נמוכה יותר. כדי להבטיח בקרה משותפת סנכרון יעיל כוללת ניטור OD 600 ומדידת הרמות של חלבון CTRA על ידי כתם מערבי בנקודות זמן מחזור התא נפרדות. OD 600 צריך להגדיל בכ 2 לקפל במהלך מחזור התא (איור 2). הכתם המערבי לרגולטור הראשי מחזור התא CTRA הוא שליטה שימושית כדי לוודא תיאום טוב (איור 2). CTRA מנוצל בתא swarmer לחסום שכפול הדנ&quot;א ונפגע על תחילתה של DNA שכפול 10. CTRA אז מסונתז בהמשך מחזור התא ומפעיל שעתוק של מארח של גני התפתחותיים הכוללים רכיבים רבים של השוטון 11,12. תיאום מוצלח יהיה לי tדפוס נדנודו של רמות חלבון CTRA. <img alt="איור 1" src="/files/ftp_upload/52633/52633fig1.jpg" /> איור 1. מחזור התא של crescentus Caulobacter. (א) סכמטי קריקטורה של מחזור תא swarmer. תאי swarmer להתמיין לתאים ארבו מוסמכים שכפול על ידי חוזר בי פילים, ולהוציא שוטונים, וייזום שכפול הדנ&quot;א. החוגים ותטא מבנים מוצגים בתוך קווי המתאר התא מייצגים כרומוזומים שקטים ומשכפלים. תאים אז להתקדם דרך מחזור תא בניית השוטון אחת בקוטב נגדי הגבעול. על החלוקה, שני סוגים ייחודיים של תאים נוצרים, שכפול חסום ניע swarmer תא ותא נייח מוסמך שכפול ארב. (ב) תוצאות עבור נציג צנטריפוגה צפיפות. צפיפות נמוכה יותר ארבה וpredivתאי isional לצוף בחלק העליון של השיפוע בזמן צפוף swarmer תאים בסופו של לכיוון החלק התחתון של הצינור. ברים סולם הם מיקרומטר 1 בתמונות מיקרוסקופי שלב. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> <img alt="איור 2" src="/files/ftp_upload/52633/52633fig2.jpg" /> איור 2. תוצאות עבור נציג מוצלח swarmer תיאום תא. מסת התאים שנמדדה על ידי OD 600 צריכה להגדיל לאט ובסופו של דבר להכפיל במהלך הקורס של assay. 1 מיליליטר aliquots היה pipetted לcuvettes פלסטיק מתיאום תא גדול וOD 600 נמדד באמצעות ספקטרופוטומטר בנקודות זמן המצוין. בנוסף, חלבון אדון מחזור תא CTRA הרגולציה צריך להיות נוכח בתא swarmer לחסום שכפול הדנ&quot;א, ואחרי השפלה מהירהביחד עם החניכה של שכפול הדנ&quot;א. CTRA לאחר מכן מחדש מאוחר יותר במחזור התא כדי להפעיל ביטוי של גנים רבים התפתחותיים כוללים רכיבי flagellar ופילים קריטיים. כתמים מערביים לאדון מחזור תא חלבון רגולטורי CTRA בוצעו על ידי לקיחת 1 aliquots מיליליטר של תיאום בקנה מידה גדולה. התאים היו resuspended ב250 μl חיץ מדגם Laemmlli לOD 600, מופרדים על 10% עמוד טריס-גלאי, הועבר לPVDF, ומחקו עם נוגדן אנטי CTRA. נהלי תיאום נכשל להוביל לכתמים מערביים CTRA ללא שינוי ברמות חלבון. נוגדן α-CTRA 12 הודגרה בדילול 1: 10,000 תמורת 1.5 שעות ב -3% TBST חלב ורחץ 3 פעמים בTBST. העז-α-ארנב המשני אז נוספה ב1: 10,000 דילול בחלב 3% TBST עבור שעה 1, שטף 3 פעמים עם TBST, וצלם בסרט באמצעות ערכת זיהוי chemiluminescent. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">אנא</a>לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> Na 2 4 HPO </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 גרם </td></tr><tr><td> H 2 O </td><td> Resuspend ב 1 L &amp; החיטוי </td></tr></tbody></table> טבלת 1. מתכון מלחים M2 20X. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> מלחים M2 20X </td><td> 50 מיליליטר </td><td></td></tr><tr><td> 0.5 M MgSO 4 </td><td> 1 מיליליטר </td><td></td></tr><tr><td> גלוקוז 20% </td><td> 10 מיליליטר </td><td> תחליף לH 2 O בM2 </td></tr><tr><td> ברזלי סולפט Chelate פתרון </td><td> 1 מיליליטר </td><td></td></tr><tr><td> 0.1 M CaCl 2 </td><td> 5 מיליליטר </td><td> הוספה האחרונה באבממטרים OID </td></tr><tr><td> H 2 O </td><td> למלא עד 1 ליטר </td><td></td></tr><tr><td> סינון סטרילי עם מסנן 0.22 מיקרומטר </td><td></td><td></td></tr></tbody></table> הטבלה 2. M2 ומתכון M2G.

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.

סנכרון של<em&gt; Caulobacter crescentus</em&gt; לחקירה של מחזור תא החיידק

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 Views.  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.

לימוד מחזור גנים ביטוי מוסדר על ידי שני פרוטוקולי סינכרון התא

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

Genetics

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.

תא הדמיה חיה של Bacillus subtilis ו Streptococcus pneumoniae באמצעות מיקרוסקופית אוטומטי זמן לשגות

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

Immunology and Infection

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.

קרינה פלואורסצנטית Live-תא הדמיה של שלמה וגטטיבי תא מחזור של חיידק חברתית גידול איטי 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 ...

Biochemistry

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.

מדידה רציפה של רעש ביולוגי ב Escherichia קולי באמצעות מיקרוסקופיה לשגות זמן

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

Bioengineering

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.

מיקרוסקופ הקרינה הפלואורסצנטית החיה כדי לחקור את הלוקליזציה של חלבון Subcellular ושינויי מורפולוגיה של תאים בחיידקים

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.

מעקב זמני של התקדמות תא מחזור שימוש cytometry הזרימה ללא הצורך בסנכרון

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

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.

חיים התא מיקרוסקופ פלורסצנטיות לבחון תהליכים חיוניים במהלך צמיחת תאים מיקרוביאלי

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.

שילוב תא Mitotic סינכרון ברזולוציה גבוהה מיקרוסקופיה קונפוקלית ללמוד את התפקיד של מחזור התא רב תכליתיים חלבונים במהלך מיטוזה

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.

ניתוח Microfluidic תא בודד של 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.

הקרנת מיקרו לייזר לחקר גיוס DNA במהלך שלב 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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