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

1.准备幼虫<ol><li>为了分析运动或立场偏好所需的幼虫，在标准条件下生长的幼虫到所需的年龄标准使用飞食品11至10测定。 </li><li>请通过拉伸丝印级尼龙网在漏斗网状过滤器。固定网在漏斗颈部橡皮筋。放置在一个烧杯中的漏斗。 </li><li>为了收集幼虫进行分析，舀一铲满包含从培养瓶觅食幼虫的食物，并用RT自来水冲洗过的筛网过滤，收集直接个人幼虫网用画笔10。 </li></ol> 2.准备测定管<ol><li>要准备堵塞试管，精心熬了4％琼脂凝胶倒入培养皿中，以1.5厘米的深度。这将提供一个插头用于管的任一侧时动物被封闭。在4％的溶液是足够致密以防止由对幼虫enetrating插头。 </li><li>确保管是干净，清晰之前，插入幼虫。如果不是，这可能会阻止从被记录的动作。 </li><li>以保证幼虫有足够的水分运动，制备含有少量的热水挤压瓶。倒置的酒瓶，仔细定位走向水槽出口和压榨轻轻地从摄像管排出的水。 </li><li>将瓶子口进入测试管。挤压瓶子提供水汽进入测试管，直到薄层冷凝出现在管的壁。视频1示出了幼虫移动在管与水分的适当的量。 </li><li>以密封幼虫在管，取出从培养皿1.5厘米厚的琼脂凝胶，并放置在一网状表面，以允许气流琼脂下方以使得琼脂塞可插入到管。按该测定管的一端插入的凝胶两次，以插入两个插头凝胶成一端。 </li><li>随着paintb匆忙，将一个幼虫进入管约1.5英寸深，按最近的幼虫年底到琼脂凝胶密封管。所产生的压力将迫使出琼脂凝胶的第二插头从相对端和在管内密封该动物。 </li><li>地方管在MB5多波束果蝇活动监视器（DAM）装置，并调整管&#39;的位置，以使动物不能超越传感器的范围内。 </li><li>为了确保测定管在运输过程中不滑出设备的红外线读取帧时，通过紧固油灰的环每个管在此其接触记录装置周围将灯管保持到位。 </li></ol> 3.测量活动<ol><li>在培养箱中记录活动，以防止可能由于阴影，荧光灯或温度变化在实验室发生的不准确的读数。参见图5为系统的一个建议的布置。 </li><li>设置一个孵化至20℃（ 见图1）。为了避免虚假记载，由于从白炽灯光源的干扰录制过程中，关掉日光灯灯光孵化器，并使用单独的LED光源，同时记录在孵化器。没有任何动物进行试验，以确保光线条件并不异常触发的传感器。应该有这个测试后无记录的移动数据。 </li><li>让幼虫以适应到20℃培养箱设置开始试验前5分钟。 </li><li>要设置DAM系统以所需记录间隔（ 如 1分钟），确保大坝系统录音软件下载到电脑主机12和前打开连接到录音室到PSIU接口DAM系统文件。 </li><li>设置所需的记录频率数据将被保存，选择首选项，然后单击上方或读取间隔选项下方，选择不同的时间框架中的数据将被存储。例如，选择阅读的间隔时间从1秒到1小时。 </li><li>要选择不同的参数记录数据，选择首选项，然后选择相应的框中下输出数据类型（计数，移动，位置和纠缠，见表1）。每个参数提供在管内的幼虫活性的独特的分析。 </li><li>要开始录制，六硼化钙用电话线将显示器连接到电源接口单元（PSIU）。该PSIU连接到电源插座。绿灯会显示相应的连接。 </li><li>通过通用串行总线（USB）电缆到Macintosh或Windows PC进行数据记录连接PSIU。在电脑上打开DAM SystemMB1v6x程序自动开始记录数据。 </li><li>后所需采集时间完成后，选择退出，那么现在退出DAM活动屏幕上;数据将在DAM系统数据自动保存。借此数据文件（ 例如 ，监控1）并拖动<html>到一个单独的文件夹。这将从坝软件存储原始数据，使他们可以在以后进行处理。 </li></ol> 4.准备用于数据处理（DAM FileScan） <ol><li>为了处理原始数据转换成可以理解的格式打开DAM FileScan应用程序并选择选择输入数据的文件夹。然后选择数据文件夹和所需文件，并选择扫描选项。最后选择的bin长到所需的读取周期。这将原始数据组织成由操作者设定的参数，程序将报告收集在该特定范围内的数据。 </li><li>选择输出的数据类型来分析所需要的运动设定（监控计数，移动，住）。下的额外读数菜单选择总和成箱（这是如果仓长度大于所选择的系统的读取间隔）。 </li><li>适当命名的文件并保存。该文件将被存储在相同的位置，从步骤3.9的文件夹。 </li></ol>_title“&gt; 5。访问数据的分析<ol><li>为了收集平均移动，过程从第4步生成为了生成一个电子表格程序表中的原始数据，打开以前保存的文件（步骤4.3），并显示文本导入向导窗口时，选择完成打开在电子表格格式的数据文件。 注：根据分析的数据类型，电子表格将被读取不同。通常情况下测量，研究的时间段将数字记录监视器移动或计数，而每一个人的阅读管会收到一个指定的字母。当在电子表格中查看数据，列KZ表示用于分别测定管1-16的槽。该行表示所收集的数据点。 </li><li>例如，如果收集在1分钟的间隔20分钟的移动，平均的20行来计算移动/分钟等测量的平均数目。 </li></ol>

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The authors declare that they have no competing financial interests.

果蝇幼虫的活性是由多种因素，包括基因型8,13年龄和环境温度2的影响。虽然能够非常详细的分析，强大的录像制品的方法已经开发了那些谁学习运动5，这种详细程度可能是多余的为那些谁希望确定活动的基本参数。这里介绍的方法采用的设备是许多实验室使用，操作方便，产生高重现性的结果，即使是对于那些主要的研究重点是不运动管理。的例子的结果表明，...

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.

图1示出了从控制第三龄幼虫的温度下反应的研究中，瓦特1118的结果，使用的监控装置，以检测在七个不同的温度下在幼虫运动的差异。幼虫洗涤并如上所述放入DAM活动装置，并放置在培养箱设置为所需的温度。该装置然后使其适应环境5分钟记录开始之前。每个幼虫单独为运动在20分钟内和每分钟移动平均数分析，计算为每一个动物，平均为每组32只动物。数据进行分析，并?...

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.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

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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Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K 次观看.  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.

视频: 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.

在果蝇幼虫中枢神经系统的剖析

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

科研

生物学

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.

果蝇幼虫NMJ夹层

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.

果蝇幼虫NMJ免疫

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.

夹层和染色果蝇幼虫卵巢

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.

醋酸形成醋酸酶激酶活性的直接检测

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.

含量测定的LRRK2的激酶活性在体外</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.

寿命测量<em&gt;果蝇</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.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

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.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

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.

体内生物传感器跟踪非凋亡胱天蛋白酶的活动在果蝇

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