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

幼虫の調製<ol><li>移動や位置の好みのための所望の幼虫を分析するために、標準的なフライ食品11を使用して10を分析したい年齢に標準的な条件下で幼虫を育てます。 </li><li>漏斗の上にシルクスクリーングレードのナイロンメッシュを延伸してメッシュフィルタを作成します。ゴムバンド付きファンネルネックでメッシュを固定します。ビーカーに漏斗を配置します。 </li><li>分析のための幼虫を収集するには、へらフル培養瓶から採餌幼虫を含有する食品のスクープと絵筆10とメッシュから直接個々の幼虫を収集し、メッシュフィルター上でRTの水道水で洗います。 </li></ol>アッセイチューブの調製<ol><li>アッセイチューブを接続するために準備するために、慎重に4％寒天ゲルを沸騰さ1.5センチの深さにシャーレに注ぎます。動物が封入されている場合に、チューブの両側のためのプラグインを提供します。 4％溶液は、pから幼虫を防止するために十分に密ですプラグをenetrating。 </li><li>チューブは前幼虫の挿入にクリーンでクリアされていることを確認してください。そうでない場合、これは記録されないような動きを阻止することができます。 </li><li>幼虫が移動するのに十分な水分を持っていることを確認するために、少量のお湯を含むスクイーズボトルを準備します。シンクに向かう出口を向けると、撮像管から水を排出するために静かに絞る慎重に、ボトルを反転。 </li><li>アッセイチューブにボトルの出口を挿入します。結露の薄い層がチューブの壁に表示さ​​れるまで、アッセイチューブに水蒸気を提供するために、ボトルを絞ります。ビデオ1は、水分の適切な量のチューブに移動する幼虫を示しています。 </li><li>チューブ内の幼虫を密封するために、ペトリ皿から1.5センチメートル厚い寒天ゲルを除去し、寒天プラグをチューブの中に挿入することができるように、寒天の下に空気の流れを可能にするために、メッシュ表面の上に置きます。一方の端部にゲルの2つのプラグを挿入するために、二回ゲルにアッセイチューブの一方の端を押してください。 </li><li> paintbとラッシュ、約1.5インチの深さのチューブに1幼虫を置き、寒天ゲル内に幼虫に最も近い端部を押してチューブをシール。得られた圧力は反対側の端部からの寒天ゲルの第2のプラグを強制し、チューブ内に動物をシールします。 </li><li>プレイスMB5マルチビームショウジョウバエアクティビティモニタ（DAM）装置で、チューブ、および動物がセンサーの範囲を超えて移動できないように、チューブのポジションを調整します。 </li><li>そのアッセイチューブは、輸送中にデバイスの赤外線読み取り枠の外にスライドさせないようにするには、それが接触する記録装置を各チューブの周りにパテのリングを固定することにより、所定の位置にチューブを保持します。 </li></ol> 3.測定アクティビティ<ol><li>インキュベータ内のレコード活性は、影、蛍光灯やラボ内の温度変化に発生することがあり、不正確な読み取りを防止します。システムの推奨構成については、図5を参照してください。 </li><li> 20°C（ 図1参照）にインキュベーターを設定します。記録時に白熱光源からの干渉による誤記録を回避するために、蛍光インキュベーターライトをオフにし、インキュベーター内で記録しながら個別のLED光源を使用しています。光の状態が異常にセンサーを誘発しないことを確実にするために、任意の動物なしで裁判を行います。この試験の後に記録された運動データがあってはなりません。 </li><li>幼虫は、アッセイ開始前の5分間20℃のインキュベーターの設定に順応することができます。 </li><li>所望の記録間隔（ 例えば、1分）にDAMシステムを設定するには、DAMシステムレコーディングソフトウェアをコンピュータ12をホストし、PSIUインターフェイスに記録チャンバーを接続する前にDAMシステム·ファイルを開くためにダウンロードされることを確認します。 </li><li> 、データが保存される時に所望の記録周波数を設定し、選択優先し、次にそのデータを様々な時間枠を選択するための読書間隔オプションの上または下をクリックし保存されます。例えば、1時間に1秒からインターバル時間を読んで選択します。 </li><li> 、データの記録に変化するパラメータを選択するプリファレンスを選択し、出力データタイプの下に対応するボックスを選択するには（カウント、移動、位置と住む、表1を参照）。各パラメータは、チューブ内の幼虫の活動のユニークな分析を提供します。 </li><li>録音を開始するには、CAB6の電話ケーブルを使用して電源インタフェースユニット（PSIU）にモニタを接続します。電源コンセントにPSIUを接続します。緑色のライトは、適切な接続を示します。 </li><li>データ記録用のMacintoshまたはWindows PCにUSB（Universal Serial Bus）ケーブルを介してPSIUを接続します。自動的にデータの記録を開始するには、コンピュータのDAM SystemMB1v6xプログラムを開きます。 </li><li>所望の収集時間が完了すると、終了を選択し、その後DAMアクティビティ画面に現在終了。データは、自動的にDAMシステムデータで保存されます。このデータファイル（ 例えば 、1を監視）し、ドラッグしてください<html>別のフォルダに。彼らは、後で処理できるようにこれは、DAMのソフトウェアからの生データを格納します。 </li></ol>処理のためのデータの準備4.（DAMファイルスキャン） <ol><li>理解しやすい形式に生データを処理するには、DAMファイルスキャンアプリケーションを開き、選択入力データフォルダを選択します。そして、データフォルダー、目的のファイルを選択して、スキャンオプションを選択します。最後に、所望の読み出し期間にビンの長さを選択します。これは、オペレータが設定したパラメータに生データを整理しますと、プログラムは、その特定の範囲内で収集されたデータを報告します。 </li><li>所望の動作設定（モニターの数、移動、住む）を分析するために、出力データ型を選択します。追加の測定値メニューの下で、ビンに選択合計（これはビンの長さが選択したシステムの読み取り間隔よりも大きい場合）。 </li><li>適切にファイルに名前を付けて保存します。ファイルには、ステップ3.9からのフォルダと同じ場所に保存されます。 </li></ol>_title &quot;&gt; 5。分析のためのデータへのアクセス<ol><li>以前に保存したファイル（ステップ4.3）を開き、平均移動、プロセススプレッドシートプログラムでテーブルを生成するには、手順4から生成された生データを収集するために、テキストインポートウィザード]ウィンドウが表示されたら、[完了]を選択し、開くためには、スプレッドシート形式のデータファイル。 注：分析されたデータの種類に応じて、スプレッドシートは、異なって読み出されます。各個別の読書チューブが指定手紙を受け取ることになりますしながら、典型的には、モニタを移動または数を測定するために、研究の期間は、数値的に記録されます。スプレッドシートのデータを表示すると、列はそれぞれ、アッセイチューブ1〜16用のスロットを表すKZ。行は、収集されたデータ点を表します。 </li><li>移動は、1分間隔で20分間採取した場合、例えば、移動/分、他の測定値の平均値を計算するために20行を平均します。 </li></ol>

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The authors declare that they have no competing financial interests.

ショウジョウバエ幼虫の活性は遺伝子型8、13歳、周囲温度2を含む様々な要因によって影響されます。非常に詳細な分析が可能な強力なビデオグラフィック方法は歩行5を研究者たちによって開発されているが、このレベルの詳細は、活動の基本的なパラメータを決定したい人のための余分な場合があります。ここで説明する方法は、多くの研究室で利用可能で?...

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.

図1は、7つの異なる温度での幼虫の運動の違いを検出するための監視装置を使用し、1118ワット、制御3齢幼虫の温度応答試験の結果を示します。幼虫を洗浄し、上記のようにDAM活動装置に入れ、所望の温度に設定したインキュベーターに入れました。記録が開始される前に装置を5分間環境に順応させました。各幼虫を個別に20分間かけて移動するために分析し、毎分移動?...

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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.

幼虫活性の測定<em&gt;ショウジョウバエ</em&gt;アクティビティモニタ

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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Research

JoVE Journal

Biology

Measurement of Larval Activity in the Drosophila Activity Monitor

9.3K ビュー.  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.

ビデオ: 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.

キイロショウジョウバエの幼虫中枢神経系の解剖

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

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.

ショウジョウバエ幼虫NMJの解剖

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.

ショウジョウバエ幼虫NMJ免疫組織化学

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

幼虫のモニタリングの心機能キイロショウジョウバエ生理学的研究のための

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

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.

の解剖と染色ショウジョウバエ幼虫卵巣

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

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.

LRRK2のキナーゼ活性を測定する in vitroで</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.

における寿命の測定<em&gt;キイロショウジョウバエ</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.

の透過方法<em&gt;ショウジョウバエ</em小分子の活性のアッセイのための&gt;胚

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.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

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.

インビボでのバイオセンサーは、 ショウジョウバエの非アポトーシスカスパーゼ活性を追跡します

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