This work was supported by NIH P20GM103643 to I. Meng.

1. הכנת הזחלים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לנתח זחל רצוי להעדפת תנועה או עמדה, לגדול זחלים בתנאים סטנדרטיים לגיל הרצוי לassay 10 באמצעות מזון זבוב סטנדרטי 11. </li><li style=";text-align:right;direction:rtl"> הפוך מסנן רשת על ידי מתיחת ניילון כיתה משי הרשת מעל משפך. אבטח את הרשת בצוואר המשפך עם גומייה. מניחים את המשפך בכוס. </li><li style=";text-align:right;direction:rtl"> לאסוף זחלים לניתוח, לגרוף מרית מלאה של מזון המכיל זחלי ליקוט מבקבוק התרבות ולשטוף במים ברז RT על מסנן הרשת, איסוף זחלים בודדים ישירות מהרשת עם מכחול 10. </li></ol> 2. הכנת Assay צינורות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להכין לחבר צינורות assay, להרתיח בזהירות ג&#39;ל אגר 4% ויוצקים לתוך צלחת פטרי לעומק של 1.5 סנטימטר. זה יספק התוספת לכל צד של הצינור כאשר חיה מוקפת. פתרון 4% הוא צפוף מספיק כדי למנוע מזחלי עמ &#39;enetrating התקעים. </li><li style=";text-align:right;direction:rtl"> ודא שצינורות נקיים וברור לפני הכניסה של זחל. אם הם לא, זה עלול לחסום תנועות ממוקלטות. </li><li style=";text-align:right;direction:rtl"> כדי להבטיח לי זחלי לחות מספקת לתנועה, להכין בקבוק לחיץ המכיל כמות קטנה של מים חמים. הפוך את הבקבוק, כוון את היציאה לכיוון כיור בזהירות ולסחוט בעדינות כדי לגרש מים מצינור האיסוף. </li><li style=";text-align:right;direction:rtl"> הכנס את שקע הבקבוק לתוך צינור assay. לסחוט את הבקבוק כדי לספק אדי מים לתוך צינור assay עד שכבה דקה של עיבוי מופיעה על הקיר של הצינור. 1 וידאו מראה זחל נע בצינור עם כמות מתאימה של לחות. </li><li style=";text-align:right;direction:rtl"> לאטום את הזחל בצינור, להסיר את הג&#39;ל אגר 1.5 סנטימטרים עובי מצלחת פטרי ומניח על משטח רשת כדי לאפשר זרימת אוויר מתחת לאגרתי כך שתקע אגר יכול להיות מוכנס לתוך הצינור. לחץ קצה אחד של צינור assay לתוך הג&#39;ל פעמיים כדי להכניס שני תקעים של ג&#39;ל לקצה אחד. </li><li style=";text-align:right;direction:rtl"> עם paintbלמהר, למקם את הזחל אחד לתוך הצינור כ 1.5 אינץ &#39;עמוק ולאטום את הצינור על ידי לחיצה על הסוף קרוב הזחל לתוך הג&#39;ל אגר. הלחץ וכתוצאה מכך יאלץ את התקע השני של ג&#39;ל אגר מהקצה השני ולאטום את החיה בתוך הצינור. </li><li style=";text-align:right;direction:rtl"> צינורות מקום במכשיר MB5 Multi-Beam תסיסנית פעילות הצג (DAM), ולהתאים את עמדות הצינורות כך שבעלי החיים לא יכולים להתקדם מעבר לטווח של החיישנים. </li><li style=";text-align:right;direction:rtl"> כדי להבטיח assay שצינורות לא להחליק מחוץ למסגרת קריאת אינפרא אדום של המכשיר במהלך הובלה, להחזיק את השפופרת במקום על ידי חיזוק טבעת של מרק סביב צינור אחד שבו אנשי קשר במכשיר ההקלטה. </li></ol> פעילות 3. מדידה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שיא פעילות בחממה כדי למנוע קריאות לא מדויקות שעלולות להתרחש כתוצאה מצללים, אורות ניאון או לשינויים בטמפרטורה במעבדה. ראה איור 5 להסדר מוצע של המערכת. </li><li style=";text-align:right;direction:rtl"> הגדר חממה עד 20 ° C (ראה איור 1). כדי להימנע מהקלטות שווא בשל הפרעות ממקורות אור הליבון במהלך הקלטה, לכבות את האורות חממת ניאון ולהשתמש במקור אור LED נפרד בעת ההקלטה בחממה. לבצע ניסוי ללא כל בעלי חיים על מנת להבטיח שתנאי התאורה לא aberrantly לעורר חיישנים. לא צריך להיות שום נתונים תנועה נרשמו לאחר בדיקה זו. </li><li style=";text-align:right;direction:rtl"> לאפשר זחלים להסתגל לטמפרטורה של 20 מעלות הגדרות C חממה ל5 דקות לפני תחילת assay. </li><li style=";text-align:right;direction:rtl"> כדי להגדיר את מערכת DAM למרווחי הקלטה רצויה (למשל, 1 דקות), להבטיח תוכנת הקלטת מערכת סכר היא הורידה לארח מחשב 12 ולפתוח את קובץ מערכת DAM לפני החיבור לתא ההקלטה לממשק PSIU. </li><li style=";text-align:right;direction:rtl"> כדי להגדיר את התדירות הרצויה ההקלטה שבה נתונים יישמרו, בחרו העדפות ולאחר מכן לחץ על מעל או מתחת לאפשרות מרווח הקריאה לבחירת מסגרות זמן שונה שבו נתוניםיאוחסן. לדוגמא, בחר לקרוא פעמים מרווח משניות 1 עד שעה 1. </li><li style=";text-align:right;direction:rtl"> כדי לבחור פרמטר שונה לנתונים הקלטה, לבחור את העדפות ולאחר מכן בחר את התיבות המתאימות בסוג נתוני פלט (ספירה, מהלכים, עמדות ולהתעכב, ראה טבלה 1). כל פרמטר מספק ניתוח ייחודי של פעילות הזחל בתוך הצינור. </li><li style=";text-align:right;direction:rtl"> כדי להתחיל בהקלטה, חבר את הצג לאספקת חשמל ממשק היחידה (PSIU) באמצעות כבל טלפון CAB6. חבר את PSIU לשקע חשמל. אור ירוק יציין חיבור מתאים. </li><li style=";text-align:right;direction:rtl"> חבר את PSIU באמצעות כבל אפיק טורי אוניברסלי (USB) למקינטוש או Windows PC להקלטת נתונים. פתח את תכנית DAM SystemMB1v6x במחשב כדי להתחיל בהקלטת נתונים באופן אוטומטי. </li><li style=";text-align:right;direction:rtl"> לאחר זמן האוסף הרצוי הוא שלם, בחר צא, ולאחר מכן צא עכשיו על מסך פעילות DAM; הנתונים לאחר מכן ניתן יהיו נשמרים באופן אוטומטי תחת מערכת נתונים DAM. קח קובץ נתונים זה (למשל, לעקוב אחר 1) וגרור<html> לתוך תיקייה נפרדת. זה יהיה לאחסן את הנתונים הגולמיים מהתוכנה DAM, כך שהם יכולים מאוחר יותר להיות מעובד. </li></ol> 4. הכנת נתונים לעיבוד (DAM FileScan) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לעבד את המידע הגולמי לפורמט מובן לפתוח את יישום DAM FileScan ובחר תיקיית נתוני קלט בחר. לאחר מכן בחר את תיקיית הנתונים ואת הקובץ הרצוי, ובחר את אפשרות הסריקה. לבסוף בחר באורך בן לתקופה הקריאה הרצויה. זה יהיה לארגן את המידע הגולמי לפרמטרים שנקבעו על ידי המפעיל והתכנית תדווח את הנתונים שנאספו בטווח מסוים. </li><li style=";text-align:right;direction:rtl"> בחר את סוג נתוני תפוקה לנתח את הגדרת התנועה הרצויה (ספירות צג, מהלכים, תשכנו). בתפריט קריאה נוספת, סכום בחר לפח (זה אם אורך בן גדול ממרווח קריאת המערכת שנבחר). </li><li style=";text-align:right;direction:rtl"> שם הקובץ כראוי ולשמור. הקובץ יישמר באותו המיקום כמו התיקייה מצעד 3.9. </li></ol>_כותרת &quot;&gt; 5. גישה לנתונים לניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לאסוף מהלכים ממוצע, תהליך הנתונים הגולמיים המופקים מצעד 4. כדי ליצור טבלה בתוכנת גיליונות אלקטרוניים, לפתוח את הקובץ שנשמר בעבר (שלב 4.3), וכאשר חלון אשף יבוא הטקסט מופיע, גימור בחר לפתוח קבצי נתונים בפורמט גיליון אלקטרוני. הערה: בהתאם לסוג הנתונים מנותח, הגיליון האלקטרוני יהיה לקרוא בצורה שונה. בדרך כלל למדידת מהלכי צג או ספירה, תקופת המחקר הזמן תירשם מספרית, בזמן שכל צינור קריאה בודד יקבל מכתב מיועד. בעת הצגת הנתונים בגיליון האלקטרוני, עמודות KZ מייצגות את החריצים לצינורות assay 1-16 בהתאמה. השורות מייצגות את נקודות נתונים שנאספו. </li><li style=";text-align:right;direction:rtl"> לדוגמא, אם מהלכים נאספו במשך 20 דקות במרווחי 1 דקות, בממוצע 20 השורות לחישוב מספר ממוצע של מהלכים / דקות ומדידות אחרות. </li></ol>

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The authors declare that they have no competing financial interests.

פעילות של זחלים תסיסנית מושפעת על ידי מגוון של גורמים, כולל גנוטיפ 8, גיל 13 וסביבת טמפרטורה 2. למרות ששיטות videographic חזקות מסוגלים ניתוח מפורט ביותר שפותחו על ידי אלה שלומדים תנועה 5, זה ברמה של פרט עשויה להיות מיותרת למי שרוצה לקבוע פרמטרים ב?...

The use of Drosophila as a genetic tool has transformed scientific knowledge of biological systems. Drosophila larvae have been used in a variety of studies including nociception1, development2 and as a model for the study of human disease genes3. Drosophila activity encompasses a range of behaviors that vary under different conditions including temperatures2, exposure to drugs4 and amongst different genotypes. Yet, despite the significant use of the larva as a model organism, a simple, standardized method to analyze larval activity has not been available. Presently, many larval locomotion studies employ sophisticated video analysis software5. While powerful, the complexity of such automated tools may discourage labs that are not already equipped to study locomotion from including analysis of informative activity parameters in their studies. In other current non-automated methods, such as the grid crawling analysis, motion is scored by a human observer, which introduces the possibility of subjectivity and limits throughput to one animal at a time6-7. A similar study used a 5-lane crawling assay, which measured the time it took larvae to travel a certain linear distance8. In such non-automated assays, displacement is measured but this does not account for non-linear travel between the start and end points. As discussed below, the method described here accounts for more of the actual larval movement, is objective, easy to operate, and offers robust throughput.
To easily study larval activity behavior without the compromise of accuracy, efficiency, or cost, this method employs the TriKinetics Drosophila Activity Monitor (DAM), a device often used to study adult activity. Using one device to study both adult and larval activity is cost-effective, and allows direct comparison of motion by animals at these two life stages. The system, featuring the manufacturer&#8217;s highest level of resolution, makes use of 17 infrared detection beams per assay tube, which record larval activity when sensor beams are broken within the 16 individual tubes. The system then automatically saves recorded information to a computer, making it available for manipulation with basic graphing software. The data obtained represents the beams that were broken by individual larvae (which can be converted into a rate), movement when a larva stays within a detector beam and the position of the animals within the assay chambers during a recording period (allowing one to calculate position preference). The system is efficient and relatively simple to operate, and brings highly reproducible basic activity analysis within the reach of any laboratory studying Drosophila larvae.
To demonstrate the power of this assay, data are presented that show its use to verify differences in activity resulting from varying ambient temperatures, as well as through the comparison of a mutant previously described as hypoactive (iav1)9 with a widely analyzed control (w1118).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila Activity Monitor, Multibeam, 16 tubes, including wires</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>MB5</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply Interface for Activity Monitor&#160;</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>PSIU24</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass 80 x 5 mm tubes for Activity Monitor (100)</td>
			<td>TriKinetics Inc.&#160;</td>
			<td>PGT 5x80</td>
			<td></td>
		</tr>
		<tr>
			<td>DAMsystemMB1v6x Data Acquisitions Software for Macintonsh OSX (Intel)</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>DAMFileScan 108x software for Macintosh</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>USB software (PSIUdrivers.zip).</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>DAMSystem Notes 308</td>
			<td><a href="http://www.trikinetics.com/">www.trikinetics.com</a></td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr>
			<td>Zeiss Stemi 2000C- Stereo Microscope</td>
			<td>Spectra Services</td>
			<td>SP-STEMI2000C-BS</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Dioxide</td>
			<td>Maine Oxy</td>
			<td></td>
			<td>anaesthesia</td>
		</tr>
		<tr>
			<td>Fly Pad</td>
			<td>Genesee</td>
			<td>59-114</td>
			<td>surface for sorting anaesthetized flies</td>
		</tr>
		<tr>
			<td>Small paint brush&#160;</td>
			<td>Winsor &#38; Newton</td>
			<td>#2 ROUND</td>
			<td>or similar, used for sorting anaesthetized flies</td>
		</tr>
		<tr>
			<td>Silk Screen Printing Mesh (160)</td>
			<td>msj-gallery.com</td>
			<td>SM160W63-3YD</td>
			<td>pore sized used in this protocol was ~ 0.1 mm</td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Genesee</td>
			<td>20-258</td>
			<td>preservative</td>
		</tr>
		<tr>
			<td>Ethanol (190proof)</td>
			<td>Pharmco</td>
			<td>111000190</td>
			<td>used to dissolve Tegosept</td>
		</tr>
		<tr>
			<td>6 oz Square Bottom Bottle (PP)</td>
			<td>Genesee</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Flugs&#34; for Plastic Fly bottles</td>
			<td>Genesee</td>
			<td>49-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Vials, Wide (PS)</td>
			<td>Genesee</td>
			<td>32-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Flugs for wide plastic vials</td>
			<td>Genesee</td>
			<td>49-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Degerminated Corn Meal</td>
			<td>Gold Medal</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila agar</td>
			<td>LabScientific</td>
			<td>FLY 8020</td>
			<td></td>
		</tr>
		<tr>
			<td>Baker&#39;s Yeast - Red Star</td>
			<td>King Arthur Flour</td>
			<td>1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated Sugar - Extra Fine</td>
			<td>Domino</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of larval activity in the drosophila activity monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

איור 1 מציג את התוצאות ממחקר תגובת טמפרטורה של זחלי instar השלישי שליטה, w 1,118, שימוש במכשיר הניטור כדי לזהות הבדלים בתנועת זחל בשבע טמפרטורות שונות. הזחלים נשטפו והניחו למכשיר פעילות DAM כפי שתוארו לעיל, והניחו בחממה להגדיר לטמפרטורה הרצויה. המנגנון היה אז ?...

Watch this Scientific Journal Video about Measurement of Larval Activity in the Drosophila Activity Monitor at JoVE.com

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

מדידה של הזחל פעילות ב<em&gt; דרוזופילה</em&gt; צג פעילות

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

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Biology

Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K Views.  University of New England. This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Video: Measurement of Larval Activity in the Drosophila Activity Monitor

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection של מערכת העצבים המרכזית הזחל ב תסיסנית melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

תסיסנית הזחל Dissection NMJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

תסיסנית הזחל NMJ אימונוהיסטוכימיה

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissection ו מכתים של תסיסנית הזחל השחלות

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

זיהוי ישיר של פעילות Acetate יוצרי של קינאז Acetate אנזימים

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Assaying פעילות קינאז של LRRK2 במבחנה</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

מדידה של תוחלת חיים ב<em&gt; דרוזופילה melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

שיטה של ​​permeabilization של<em&gt; דרוזופילה</em&gt; עוברים למבחנים של מולקולה קטנה פעילות

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

מדידה של קצב חילוף החומרים ב<em&gt; דרוזופילה</em&gt; באמצעות Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

מדידה של הזחל פעילות ב

Summary

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Chapters in this video

Dissection של מערכת העצבים המרכזית הזחל ב תסיסנית melanogaster

תסיסנית הזחל Dissection NMJ

תסיסנית הזחל NMJ אימונוהיסטוכימיה

Dissection ו מכתים של תסיסנית הזחל השחלות

זיהוי ישיר של פעילות Acetate יוצרי של קינאז Acetate אנזימים

Assaying פעילות קינאז של LRRK2 במבחנה

מדידה של תוחלת חיים ב

שיטה של permeabilization של

מדידה של קצב חילוף החומרים ב

מדידות הכינון פונקציונלי ופעילות הערוץ של מטוהרים wildtype וחלבון CFTR Mutant

מדידה של הזחל פעילות ב

Summary

Explore More Videos

Chapters in this video

Dissection של מערכת העצבים המרכזית הזחל ב תסיסנית melanogaster

תסיסנית הזחל Dissection NMJ

תסיסנית הזחל NMJ אימונוהיסטוכימיה

Dissection ו מכתים של תסיסנית הזחל השחלות

זיהוי ישיר של פעילות Acetate יוצרי של קינאז Acetate אנזימים

Assaying פעילות קינאז של LRRK2 במבחנה

מדידה של תוחלת חיים ב

שיטה של ​​permeabilization של

מדידה של קצב חילוף החומרים ב

מדידות הכינון פונקציונלי ופעילות הערוץ של מטוהרים wildtype וחלבון CFTR Mutant

שיטה של permeabilization של