Name: basic caenorhabditis elegans methods: synchronization and observation
Uploaded: 2012-06-10T14:00:00.000+00:00
Duration: 11 min 34 s
Description: Watch this Scientific Journal Video about Basic Caenorhabditis elegans Methods: Synchronization and Observation at JoVE.com

המחברים רוצים להכיר MICINN (התוכנית ועד ההורים תומכים מונסראט פורטה דה לה ריבה), AGAUR (PhD מלגה כדי לורה Fontrodona), Instituto de Salud קרלוס השלישי (תוכנית מיגל Servet תמיכה ג&#39;וליאן Cerón), ומארי קירי IRG, ISCIII ו IDIBELL למימון במעבדה.

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Biol. 694, 47-60 (2010).</li><li>Joshi, P. M., Riddle, M. R., Djabrayan, N. J., Rothman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+model+for+stem+cell+biology.">Caenorhabditis elegans as a model for stem cell biology.</a> Dev. Dyn. 239, 1539-1554 (2010).</li><li>Waters, K. A., Reinke, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+and+intrinsic+control+of+germ+cell+proliferation+in+Caenorhabditis+elegans.">Extrinsic and intrinsic control of germ cell proliferation in Caenorhabditis elegans.</a> Mol. Reprod. Dev. 78, 151-160 (2011).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> Wormbook. , (2006).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans.">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev. Biol. 8, 49 (2008).</li><li>Olahova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+redox-sensitive+peroxiredoxin+that+is+important+for+longevity+has+tissue-+and+stress-specific+roles+in+stress+resistance.">A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 19839-19344 (2008).</li><li>Voisine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+therapeutic+drugs+for+huntington's+disease+using+Caenorhabditis+elegans.">Identification of potential therapeutic drugs for huntington's disease using Caenorhabditis elegans.</a> PLoS One. 2, e504 (2007).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Aceves, J., Erlij, D., Martinez-Maranon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+paralysing+action+of+tetramisole+on+Ascaris+somatic+muscle.">The mechanism of the paralysing action of tetramisole on Ascaris somatic muscle.</a> Br. J. Pharmacol. 38, 602-607 (1970).</li><li>Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cell+biology.">Methods in cell biology.</a> Wormbook. , (2006).</li><li>Pepper, A. S., Killian, D. J., Hubbardm, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+Caenorhabditis+elegans+glp-1+mutants+suggests+receptor+interaction+or+competition.">Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition.</a> Genetics. 163 (1), 115-132 (2003).</li><li>Morley, J. F., Brignull, H. R., Weyers, J. J., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+threshold+for+polyglutamine-expansion+protein+aggregation+and+cellular+toxicity+is+dynamic+and+influenced+by+aging+in+Caenorhabditis+elegans.">The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 10417-10422 (2002).</li></ol>

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נמטודות סנכרון פתרונות הלבנת מספר תוארו. ניסינו חמישה מתכונים שונים (טבלה I) ו, בידינו, הם לא הראו הבדלים משמעותיים הסנכרון של אוכלוסיות תולעת (איור 1). עם זאת, בניסויים שלנו לא מראים פרמטרים כגון טמפרטורה (איור 2), הפתרון יחס הלבנת: נפח של תולעים (איור 3) וכן את נפח M9 עם שבו עוברים מודגרת של הבקיעה (איור 4) עושים משפיע ההישרדות של תולעים, בהיותו קשור אוורור תקין של תרבות. בתנאים שלנו רועדות, בעוד שפופרת של 15 מ&quot;ל בנפח של 1 מ&quot;ל מאפשר הישרדות של תולעים כל, בנפח של 5 מ&quot;ל כבר יותר מדי על מנת לאפשר בקיעה ביצים נאות דומה לנפח מרבי של 15 מ&quot;ל (לא מוצג) . סי אלגנס פיתוח במהלך הפיתוח שלה, <em&gt; ג elegans עובר דרך ארבעה שלבים הזחל (איור 5) לפני לשלב הבוגר. קו נבט הוא אינדיקטור טוב של השלב ההתפתחותי של C. elegans. התכונה הקלה ביותר של ג elegans פיתוח ניתן לראות תחת Nomarski אופטיקה הוא פיתוח של הפות, אשר מתחיל להיווצר בשלב מוקדם L4. בהתחלה, רק לומן קטן הוא ציין, שאחר כך מתרחב שנקרא הצורה &quot;עץ חג המולד&quot;, על ידי L4 באמצע מאוחר. בסופו של דבר, עד סוף שנת L4 הפות סוגר (איור 6). מצד שני, מכתים DAPI מאפשר התבוננות בהתפתחות בלוטת המין. מתוך ארבעת התאים L1 כדי בלוטת המין חלוקת תאים מתארך ב L2 ו L3. ב L3 התאים קצה דיסטלי ניתן לראות, החל להעביר dorsally. גם המיוזה מתחילה עד סוף L3. על קצה דיסטלי L4 תאים להגיע מעמדה הסופי שלהם בתאי הנבט לבדל את הזרע. עד סוף שנת הייצור L4 הזרע מסתיים וייצור הביצית מתחיל. ב adulטי עוברי תולעים ניתן לראות בתוך הרחם (איור 7). פיתוח טמפרטורה סי אלגנס מתפתח בקצב שונה בהתאם לטמפרטורה: אמנם זה לוקח בערך 90 שעות מרגע הטלת הביצה ועד תולעת חדש מתחיל להטיל ביצים שלה ב 15 מעלות צלזיוס, 45 שעות זה מספיק כאשר גדל במהירות של 25 ° C (איור 6). המחקר של שיעור ההפרש פיתוח בטמפרטורות שונות מוביל גמישות יחסית בהקמת תנאי וביצוע ניסויים. בנוסף, היא מציעה את האפשרות לא רק לעקוב אחר ההשפעות של טיפול מסוים או שינוי (לדוגמה אללים רגישים לטמפרטורה), אלא גם כדי ליצור את התנאים הטובים ביותר בהם מבצעת ניסוי מסוים.

basic caenorhabditis elegans methods: synchronization and observation

מחקר בביולוגיה מולקולרית התפתחותית של Caenorhabditis elegans נמטודות החלה בתחילת שנות השבעים של סידני ברנר וזה ומאז נעשה שימוש נרחב כאורגניזם מודל 1. ג elegans הוא בעל תכונות מפתח כגון שקיפות ופשטות, ועל מחזור החיים הקצר שהפכו אותה מערכת ניסיונית מתאים למחקרים ביולוגיים בסיסיים במשך שנים רבות 2. תגליות נמטודות הזה יש השלכות רחבות בגלל תהליכים תאיים ומולקולריים רבים חיים פיתוח שליטה הם 3 משומר האבולוציוני. סי אלגנס מחזור החיים עובר בשלב העוברי וארבעה שלבים הזחל לפני בעלי החיים מגיעים לבגרות. הפיתוח יכול לקחת 2 עד 4 ימים, תלוי בטמפרטורה. בכל אחד מהשלבים התכונות האופייניות כמה ניתן לצפות. הידע של השושלת תא המוחלטת 4,5 יחד עם annotat עמוקיון של הגנום שלו להפוך את נמטודות למודל רב בתחומים מגוונים כמו לנוירוביולוגיה 6, הזדקנות 7,8, ביולוגיה לתאי גזע 9 וביולוגיה נבט קו 10. תכונה נוספת שהופכת ג elegans מודל אטרקטיבי לעבוד היא האפשרות לקבל אוכלוסיות של תולעים מסונכרנים בשלב מסוים באמצעות פרוטוקול פשוט יחסית. קלות תחזוקה להפצת נמטודות זה להוסיף אפשרות של סנכרון לספק כלי רב עוצמה כדי להשיג כמויות גדולות של תולעים, אשר ניתן להשתמש בהם עבור מגוון רחב של ניסויים קטנים או תפוקה גבוהה כגון מסכי RNAi, microarrays, רצף מסיבי, immunoblot או הכלאה באתרו, בין היתר. בגלל השקיפות שלה, ג מבנים elegans ניתן להבחין תחת מיקרוסקופ באמצעות התערבות דיפרנציאלי מיקרוסקופ ניגודיות, הידוע גם בשם Nomarski MICROSלהעתיק. השימוש קלסר DNA ניאון, DAPI (4 &#39;,6-diamidino-2-phenylindole), למשל, יכול להוביל לזיהוי לוקליזציה ספציפי של תאים בודדים, כמו גם מבנים subcellular / פגמים הקשורים אליהם.

Watch this Scientific Journal Video about Basic Caenorhabditis elegans Methods: Synchronization and Observation at JoVE.com

Basic Caenorhabditis elegans Methods: Synchronization and Observation

קלות של תחזוקה להפצת נמטודות<em&gt; ג elegans</em&gt; להפוך אותו אורגניזם מודל נחמד לעבוד איתו. אפשרות של תולעים סינכרון מאפשר עבודה עם כמות משמעותית של הנושאים בשלב התפתחותי דומה, מה מאפשר לימוד של תהליך מסוים אצל בעלי חיים רבים.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

basic-caenorhabditis-elegans-methods-synchronization-and-observation

Nanyang Technological University

Research

JoVE Journal

Biology

Basic Caenorhabditis elegans Methods: Synchronization and Observation

46.4K Views.  Bellvitge Institute for Biomedical Research. קלות של תחזוקה להפצת נמטודות ג elegans להפוך אותו אורגניזם מודל נחמד לעבוד איתו. אפשרות של תולעים סינכרון מאפשר עבודה עם כמות משמעותית של הנושאים בשלב התפתחותי דומה, מה מאפשר לימוד של תהליך מסוים אצל בעלי חיים רבים.

Video: Basic Caenorhabditis elegans Methods: Synchronization and Observation

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

שיטות מתוקננת למדידת אינדוקציה של תגובת הלם חום באלבורבדיוטיס

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Environment

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

סקר שיטות בעלות נמוכה למדידת תוחלת החיים ותוחלת הבריאות ב - Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Genetics

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

ניתוח השוואתי של שיטות ניסיוניות לכימות פעילות בעלי חיים בקאנורבדיטיס אלגנס מודלים של מחלות מיטוכונדריאליות

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

Behavior

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

ניטור הצטברות חלבונים קינטיקה ב Vivo באמצעות ספירת הכללה אוטומטית ב Elegans Caenorhabditis

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biochemistry

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

מדידה של קצב הצריכה של חמצן בשלם Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

גישות כמותיים ללימוד מבנים סלולאריים ומורפולוגיה ארגונית בקנבאבדיוטיס

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

מדידות התגובות הפיזיולוגיות של הלחץ בג. אלגיה

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

מיקרו-דיסקציה של לייזר עבור יישומים של רקמה אחת אגנוסטית של מינים

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

שימוש בנתונים של תולעת בודדת כדי לכמת את ההטרוגניות באינטראקציות של Caenorhabditis elegans-Bacterial

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

<meta charset="utf8"></head><body>ניתוח מורפולוגיה מיטוכונדריאלית ב - C. elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

<meta charset="utf8"></head><body>הדמיה וכימות מורפולוגיה מיטוכונדריאלית ב - C. elegans במהלך ההזדקנות

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

הדמיה וכימות מורפולוגיה מיטוכונדריאלית ב - C. elegans במהלך ההזדקנות

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

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