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

애벌레 1. 준비 <ol><li> , 운동 또는 위치를 기본 설정으로 원하는 유충을 분석 표준 플라이 음식 (11)를 사용하여 10를 분석하기 위해 원하는 시대에 표준 조건 하에서 유충 성장합니다. </li><li> 실크 스크린 수준의 나일론 깔때기를 통해 메쉬 스트레칭으로 메쉬 필터를 확인합니다. 고무 밴드와 깔때기 목에 메쉬를 고정합니다. 비커에 깔때기를 놓습니다. </li><li> 분석을 위해 애벌레를 수집하려면, 주걱 가득 배양 병에서 구하고 유충을 포함하는 식품의 특종과 붓 10 메쉬에서 직접 개별 애벌레를 수집, 메쉬 필터를 통해 RT의 수돗물로 세척한다. </li></ol> 분석 튜브 2. 준비 <ol><li> 분석 튜브를 연결하기 위해 준비하려면 신중하게 4 % 한천 겔을 끓여 1.5 cm의 깊이에 페트리 접시에 붓는다. 동물 봉입 할 때 튜브의 양측에 대해 플러그를 제공 할 것이다. 4 % 용액 (P)에서 유충을 방지하기에 충분히 조밀플러그를 enetrating. </li><li> 튜브 깨끗하고 유충의 삽입 분명 이전에 있는지 확인합니다. 그렇지 않은 경우, 기록되는 움직임을 차단 할 수있다. </li><li> 유충 이동 충분한 수분을 보장하기 위하여 온수를 소량 함유하는 스퀴즈 병을 준비한다. 조심스럽게 싱크쪽으로 출구 방향을, 병 전환 및 픽업 관에서 물을 추방 부드럽게 쥐어 짜기. </li><li> 분석 튜브에 병 콘센트를 삽입합니다. 축합 박층 튜브의 벽에 나타날 때까지 시험 튜브에 수증기를 전달하는 스퀴즈 병. 비디오 1은 수분 적당량 관으로 이동 유충을 나타낸다. </li><li> 튜브를 밀봉 유충, 페트리 접시에서 1.5 cm 두께의 한천 겔을 제거하고 한천 플러그는 튜브 내로 삽입 될 수 있도록 한천 아래의 공기 흐름을 허용하기 위해 메시의 표면 위에 놓는다. 일 단부에 두 겔 플러그를 삽입하는 회 겔로 분석 튜브의 한쪽 끝을 누른다. </li><li> paintb으로서두 약 1.5 인치 깊이 튜브로 한 유충을 배치하고 한천 겔에 유충 가까운 단부를 눌러 튜브를 밀봉. 생성 된 압력은 대향 단부로부터 한천 겔의 제 2 플러그를 강제 튜브 내에 동물을 밀봉한다. </li><li> 장소 MB5 멀티 빔 초파리 활동 모니터 (DAM) 장치의 튜브 및 동물이 센서의 범위를 넘어 이동할 수 있도록 튜브 &#39;의 위치를 조정합니다. </li><li> 튜브 운송 중 장치의 적외선 판독 프레임 밖으로 밀어하지 않는 분석을 보장하기 위해, 각각의 튜브에 접촉 기록 장치 주위에 퍼티의 고리를 고정하여 장소에 튜브를 개최합니다. </li></ol> 3. 측정 활동 <ol><li> 인큐베이터에서 녹음 활동으로 인해 그림자, 형광등 또는 실험실에서 온도 변화로 발생할 수있는 부정확 한 측정을 방지합니다. 시스템의 제안 배치는 그림 5를 참조하십시오. </li><li> 20 ° C (그림 1 참조)에 인큐베이터를 설정합니다. 녹화 중에 인해 백열등 광원의 간섭에 허위 기록을 방지 형광 인큐베이터 조명을 끄고 인큐베이터에서 녹음하는 동안 별도의 LED 광원을 사용합니다. 비정상적으로 센서를 트리거하지 않는 조명 조건을 보장하기 위해 모든 동물없이 시험을 수행합니다. 이 테스트 이후에는 기록 된 이동 데이터가 없어야합니다. </li><li> 애벌레 분석의 시작하기 전에 5 분 동안 20 ° C 배양기 설정에 적응하도록 허용합니다. </li><li> 원하는 기록 간격 (예를 들면, 1 분)에 댐 시스템을 설정하려면 DAM 시스템 레코딩 소프트웨어는 컴퓨터 (12)를 호스팅하고 PSIU 인터페이스에 기록 챔버를 연결하기 전에 DAM 시스템 파일을 엽니 다 다운로드되어 있는지 확인합니다. </li><li> 다음 데이터가 저장 될 때의 원하는 녹화 주파수 선택 환경 설정을 설정하려면 서로 다른 시간 프레임을 선택하려면 위 또는 독서 간격 옵션 아래를 클릭하는 데이터저장됩니다. 예를 들어, 1 시간에 1 초 시간 간격에서 독서를 선택. </li><li> , 기록 데이터에 대한 다양한 매개 변수를 선택 환경 설정을 선택하고 출력 데이터 타입에 따라 해당 상자를 선택하려면 (수, 이동, 위치 및 거, 표 1 참조). 각 매개 변수는 튜브 내에서 애벌레 활동의 고유 한 분석을 제공합니다. </li><li> 녹음을 시작 CAB6 전화 케이블을 사용하여 전원 공급 장치 인터페이스 장치 (PSIU)에 모니터를 연결합니다. 전원 콘센트에 PSIU를 연결합니다. 녹색 등이 해당 연결을 나타냅니다. </li><li> 데이터 기록을위한 매킨토시 또는 Windows PC에 범용 직렬 버스 (USB) 케이블을 통해 PSIU를 연결합니다. 자동 데이터 기록을 시작하기 위해 컴퓨터에 댐 SystemMB1v6x 프로그램을 엽니 다. </li><li> 바람직한 수집 시간이 완료되면 종료를 선택한 다음 DAM 활성 ​​화면에서 지금 종료; 데이터가 자동으로 DAM 시스템 데이터 아래에 저장됩니다. 이 데이터 파일을 가지고 (예를 들어 1, 모니터)와 드래그<html> 별도의 폴더에. 그들은 나중에 처리 될 수 있도록이 DAM 소프트웨어의 원시 데이터를 저장할 것이다. </li></ol> 처리를위한 데이터 준비 (4) (DAM 파일 검사) <ol><li> 이해할 수있는 형식으로 원시 데이터를 처리하려면 댐 파일 검사 응용 프로그램을 열고 선택 입력 데이터 폴더를 선택합니다. 그런 다음 데이터 폴더와 원하는 파일을 선택하고 검색 옵션을 선택합니다. 마지막으로 원하는 독서 기간에 빈 길이를 선택합니다. 이는 작업자에 의해 설정된 파라미터에 원시 데이터를 구성되고 프로그램은 특정 범위 내에서 수집 된 데이터를보고 할 것이다. </li><li> (모니터 카운트, 이동, 거) 원하는 동작 설정을 분석하기 위해 출력 데이터 유형을 선택합니다. 여분의 판독 메뉴에서, 빈에 선택 합 (이 빈 길이가 선택한 시스템 판독 간격보다 큰 경우). </li><li> 적절하게 파일 이름을 지정하고 저장합니다. 파일은 단계 3.9에서 폴더와 같은 위치에 저장한다. </li></ol>분석을위한 _title &quot;&gt; 5. 데이터 액세스 <ol><li> 이전에 저장 한 파일 (단계 4.3)를 열고, 평균 이동, 처리 스프레드 시트 프로그램에서 테이블을 생성하려면 4 단계에서 생성 된 원시 데이터를 수집, 텍스트 가져 오기 마법사 창이 나타나면, 선택 마무리 열기 위해 스프레드 쉬트 형식의 데이터 파일. 주 : 분석 데이터 타입에 따라, 스프레드 시트는 다르게 판독 될 것이다. 각각의 독서 관이 지정 편지를 받게됩니다 동안 일반적으로, 연구의 시간이 숫자로 기록됩니다 모니터 이동 또는 수를 측정합니다. 스프레드 시트에 데이터를 볼 때, 열은 검정 튜브 1-16 각각에 대해 KZ 슬랏을 나타낸다. 행은 수집 된 데이터 지점을 나타낸다. </li><li> 이동은 1 분 간격으로 20 분 동안 수집 된 경우, 예를 들어, 이동 / 분 및 다른 측정의 평균 수를 계산하는 평균 20 행. </li></ol>

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The authors declare that they have no competing financial interests.

초파리 유충의 활동은 유전자형 8, 13 세 및 주위 온도 2를 포함한 다양한 요인에 의해 영향을 받는다. 매우 상세한 분석 할 수있는 강력한 비디오 그래픽 방법은 운동 (5)을 연구하는 사람들에 의해 개발되었지만, 세부 사항의 수준은 활동의 기본 매개 변수를 결정하고자하는 사람들을 위해 불필요 할 수있다. 여기에 기재된 방법은, 많은 실험실에서 사용할 디바이스...

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.

도 1은 상이한 온도에서 일곱 유생 운동의 차이를 검출하기 위해 모니터링 장치를 사용하여, 1118 w 제어 셋째 령 유충의 온도 반응 연구로부터의 결과를 나타낸다. 유충을 세척 및 전술 한 바와 같이 DAM 동작 장치에 배치하고, 원하는 온도로 설정 배양기에 넣었다. 녹화가 시작되기 전에 장치가 다음 5 분 동안 환경에 적응시켰다. 각 애벌레 개별적 20 분에 걸쳐 운동 분당 움직?...

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.

에서 애벌레 활동의 측정<em&gt; 초파리</em&gt; 활동 모니터

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

measurement-of-larval-activity-in-the-drosophila-activity-monitor

Research

JoVE Journal

Biology

Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K 조회수.  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.

비디오: 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.

Drosophila Melanogaster에서 애벌레 CNS의 해부

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

연구

생물학

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.

Drosophila 애벌레 NMJ의 해부

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.

Drosophila 애벌레 NMJ Immunohistochemistry

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.

해부 그리고 스테 이닝 Drosophila 애벌레 난소

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.

효소 아세테이트 키나제의 아세테이트 - 형성 활동의 직접적인 탐지

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.

LRRK2의 키나제 활동 시금 체외에서</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.

의 수명 측정<em&gt; Drosophila 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.

의 Permeabilization의 방법<em&gt; 초파리</em작은 분자 활동의 분석 실험을위한&gt; 태아

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

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.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

생체 내에서 바이오 센서는 초파리에서 비 세포 사멸 카스파 활동을 추적

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...