저자는 프로토콜을 개발하고 시약을 제공하는 인물, N. Gudimchuk 및 P. Zakharov에 대한 이미지를 제공하기 위해, N. Dashkevich, N. Gudimchuk 및 A. Korbalev을 재사용 할 수있는 유량 용기를 디자인하고 제조하는 데 도움을 위해 FI Ataullakhanov에게 감사의 말씀을 전합니다 텍스트 편집 및 팁 및 토론을위한 Grishchuk 연구소의 다른 회원들과 도움을 제닌 표지 미세 소관 씨, A. Potapenko를 준비합니다. 이 작품은에 의해 부분적으로 지원되었다 NIH 부여 GM R01-098389과 킴멜 학자 ELG 펜실베니아 근육 연구소에서 파일럿 부여, RFBR 12 채로 04-00111-13-04-40190-H를 부여하고 (13) - 04-40188-H, 과학 상임위원회 보조금 러시아 아카데미 FI Ataullakhanov에 (분자 시스템 통합 및 분자 및 분자 세포 생물학 프로그램의 메커니즘), NIH는 JR 매킨토시에 GM R01 GM033787을 부여하고, 드미트리 Zimin 왕조 재단 박사의 교제 VAV

저자가 공개하는 게 없다.

대부분의 단일 분자 분석은 오늘날 일상적으로 크게 특이 단백질의 고집을 줄이기 위해 특수 처리 커버 슬립을 사용합니다. 우리가 여기에서 설명하는 절차는 하워드 랩 32에서 개발 된 원래의 프로토콜의 변형이며, 우리는 커버 슬립을 silanizing은도 형광을 사용하지 않는 DIC 기반 비드 분석과 노력의 가치는 사실을 알게 될 것입니다. 이러한 커버 슬립 조립 챔버 훨씬 청소기 표면을 보여?...

미세 소관은 높은 세포 구조, 세포 운동성, 세포 분열, 세포 내 수송 1에 중요한 골격 구조를 보존하고 있습니다. 이러한 동적 중합체는 GTP의 존재 튜 불린에서 조립 이들은 자발적 성장이 단축 전환. 미세 소관은 (직경 25 nm의) 콘트라스트가 광학 현미경으로 미세 소관을 관찰하는 데 사용되어야합니다 강화하기 때문에 특별한 광학 기술 매우 얇은입니다. 이러한 폴리머와 이전 작업은 미분 간섭 대비 (DIC)을 3 사용하여 동적 동작을 살펴 보았다. 체외에서 이것과 유사한 연구는 일반적인 실험 조건에서 미세 소관은, 드물게 해중합에 재앙과 스위치를 거쳐야 밝혀마다 5 ~ 15 분 (이 주파수는 28 ~ 32 ° C에서 검사 7-15 밀리미터 용해 튜 불린의 농도입니다 ) 4. 다른 기술은 따라서 인덕턴스에 제안되었다제어 방식으로 전자 미세 소관 해중합. 미세 소관 쇼트닝 여기서 설명한 바와 같이, 거리 수용성 튜 불린 5,6 세척 레이저 광 (7)로 미세 소관을 절단 또는 분할 된 미세 소관 8을 사용하여 발생 될 수있다. 분할 된 미세 소관뿐만 아니라, 확률 론적으로 전환 폴리머를 사용하여 이전 작업은 이러한 염색체, 소포, 단백질 코팅 비즈 작은 세포 내화물은, 단축 미세 소관 9-13의 끝에서 이동할 수 있다는 것을 발견했다. 이러한 현상은 유사 분열 세포의 염색체 운동에 대한 직접적인 의미를 생각하고, 기본 메커니즘이 활성화 연구 14-16에서 현재됩니다. 최근, 총 내부 반사 형광 (TIRF) 현미경 등의 형광 기반 기술, 동적 인 미세 소관과 운동성을 연구하기 위해 사용되었다가 17-24로 끝났습니다. 이 방법의 장점은 허용한다는 것이다 상호 작용의 검토다른 형광 물질로 표지 단백질을 사용하여 실시간으로 미세 소관과 미세 소관 결합 단백질 사이의. 몇몇 단백질 복합체는 미세 소관 단부를 연신 및 / 또는 단축으로 processively 이동 발견되었다. 그들은 미세 소관 관련 단백질 Dam1 10,12,18, Ska1 19 일 및 XMAP215 (20)뿐만 아니라, 키네신 모터 Kif18A 21, 22, MCAK 23 CENP-E (24)이 (가) 있습니다. 이 단백질은 EB1 (25)와 같은 고전적인 팁 추적 단백질의 그것과 근본적으로 다른 processive 팁 추적을 나타낸다. EB1 분자 및 관련 파트너는 안정적으로 동적 미세 소관은 개별 분자가 빠르게 용해 수영장 (26)와 교환 만 ~ 0.8 초 미세 소관 팁에 바인딩 유지, 종료와 관련된 남아있는 것처럼 보이지만. 미세 소관이 많은 마이크론에 끝 반면, processive 팁 추적기, Dam1처럼, 여행, 및 미세 소관 조언과의 관계는 M 지속될 수모든 초. 팁 연관 시간뿐만 아니라, 추적 결과 속도는, 팁 추적 복합체를 형성하는 분자 (27)의 수에 크게 의존한다. 큰 단백질 앙상블은 일반적으로 훨씬 더 팁 추적기입니다. 예를 들어, 분리 된 효모 kinetochores와 같은 복잡한 어셈블리는 미세 소관에 결합 된 상태를 유지할 수는 시간 28 일 끝납니다. 일부 미세 소관 결합 단백질은, 예를 들어 Ndc80 동원체의 단백질 복합체, 단일 분자 수준에서 종료 미세 소관으로 추적 할 수없는 것으로, 아직 Ndc80은 구슬화물 19,29-31의 움직임을 결합에 매우 효율적입니다되었습니다. 따라서, 다른 단백질 복합체뿐만 아니라 생물학적 역할에 의한 팁 추적 메커니즘을 이해하기 위해, 그 팁 추적 복잡한 분자의 개수의 함수로서 팁 추적을 검사 할뿐만 아니라, 결정하는 것이 중요하다 구슬화물의 표면에 집단 운동을 전시하는이 단지의 기능을 제공합니다. (그림 1A)와 함께 실험을 준비하고 수행 할 수있는 세부적인 프로토콜을 제공하는 아래. 첫째, 상업적으로 이용 가능한 유리 슬라이드 짧은 폴리에틸렌 관 (프로토콜 1)를 첨부하도록 수정됩니다. 재사용 할 수있는 현미경 흐름 챔버는 다음과 같은 슬라이드에서 조립 및 화학적 또는 플라즈마 세정 및 실란 커버 슬립 (프로토콜 2) 32 ~ 34됩니다. 얻어진 챔버 체적은 유입 배관의 용적 등 만 20-25 μL (또는 15 μL 한 작게, 프로토콜 1 주 3 참조)이다. 시판 유동 챔버도 사용될 수 있지만, 그 양이 단백질의 불필요한 낭비로 이어지는, 보통 크다. 큰 챔버가 사용되는 경우, 아래의 프로토콜의 모든 솔루션의 볼륨이 비례 적으로 조정해야한다. 미세 소관의 씨앗이 서서히 가수 분해 GTP 아날로그, GMPCPP (구아노 신-5&#39;-[(α, β) methyleno] 트리 포스페이트) (프로토콜 3 등을 사용하여, 준비, 하이 먼 &lt;도 참조EM&gt; 등. 35). 씨앗 청소 커버 슬립에 고정하고 표면은 이후에 다른 단백질의 비특이적 흡착을 방지하기 위해 차단 32 (프로토콜 4 제닌 사용 씨의 고정화를 설명합니다). 분단 미세 소관은 5 프로토콜을 사용하여 제조 될 수있다. 이 방법의 주된 이론적 근거는 GTP의 존재하에 동적 미세 소관 형성 중합체, GMPCPP 포함될 안정 튜 불린 세그먼트의 짧은 &quot;캡&quot;을 추가하여 일시적으로 안정화 될 수 있다는 것이다. 이 모자는 로다 민 표지 튜 불린을 포함, 그래서 그들은 530-550 nm의 레이저 또는 수은 아크 램프 (프로토콜 6) (36)의 시야를 비추는 것만으로 간단하게 제거 할 수 있습니다. 팁 트래킹 신호의 형광 강도는 고려 현미경 야 조사 (프로토콜 12)의 요철을 가지고, 분해 미세 소관 단부로 여행 분자의 수를 추정하기 위해 사용될 수있다. 유사한 접근법이 사용될 수있다27 (프로토콜 8)에 기술 된 바와 같이 제조 된 해중합의 미세 소관 단백질 코팅 구슬 사이의 상호 작용을 연구합니다. 일부 단백질은 쉽게 분할 된 미세 소관의 벽에 결합하지만, 레이저 핀셋도하여 그 바인딩 홍보, 미세 소관 벽 근처 비드를 보유 할 수 있습니다.

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 1: Microscopy and other equipment.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss 
			Nikon</td>
			<td>Axio Imager 2 
			Eclipse Ti</td>
			<td>other microscope models capable of DIC and epi-fluorescence-imaging can be used</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss 
			Nikon</td>
			<td>420490-9900-000 
			CFI Apo 100x Oil 1.49</td>
			<td>100X, DIC, 1.3-1.49 NA</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150803, 150819-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent filter cube</td>
			<td>Chroma</td>
			<td>49004 or 49008 
			41017 or 49020</td>
			<td>optimized for Rhodamine fluorescence&#160; 
			optimized for GFP fluorescence</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>freeware MicroManager 
			Molecular Devices</td>
			<td>not applicable 
			MetaMorph 7.5</td>
			<td>http://valelab.ucsf.edu/~MM/MMwiki/ 
			other software can be used to acquire images and for a particle tracking</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3, DU-897E-cs0-#BV</td>
			<td>Highly sensitive EMCCD camera</td>
		</tr>
		<tr>
			<td>Trapping laser</td>
			<td>IPG Photonics</td>
			<td>YLR-10-1064-LP</td>
			<td>1064 nm laser, 10 W</td>
		</tr>
		<tr>
			<td>Fluorescence excitation lasers</td>
			<td>Coherent, Inc. 
			Coherent, Inc.</td>
			<td>Sapphire 488 LP 
			Sapphire 552 LP</td>
			<td>excitation of green fluorophores 
			excitation of red fluorophores</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flow chambers</td>
			<td>Warner Instruments</td>
			<td>RC-20 or RC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole Palmer 
			Harvard Apparatus</td>
			<td>Masterflex 77120-52 
			Pico Plus</td>
			<td>Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.&#160;</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 2: Microscopy chamber preparation.</td>
		</tr>
		<tr>
			<td>Modified microscope slides for reusable chambers</td>
			<td>Precision Glassblowing of Colorado</td>
			<td>Custom order www.precisionglassblowing.com</td>
			<td>Sonic slots in slides using schematics in Figure 1</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427410</td>
			<td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427400</td>
			<td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td>
		</tr>
		<tr>
			<td>Regular microscope slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Other similar slides can be used</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-150, 48366-067</td>
			<td>Other similar coverslips can be used</td>
		</tr>
		<tr>
			<td>Silicon sealant</td>
			<td>World Precision Instruments</td>
			<td>KIT, SILICON SEALANT 5 MIN CURE</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Loctite</td>
			<td>83082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Scotch 3M</td>
			<td>AD114</td>
			<td>Or cyanoacrylate adhesive from other manufacturers</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 3: Coverslips cleaning and coating.</td>
		</tr>
		<tr>
			<td>Molecular Sieves, Grade 564</td>
			<td>Macron</td>
			<td>4490-04</td>
			<td width="200px"></td>
		</tr>
		<tr>
			<td>Coverglass Staining Jar</td>
			<td>Ted Pella, Inc.</td>
			<td>21036</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Ceramic Holder</td>
			<td>Thomas Scientific</td>
			<td>8542e40</td>
			<td></td>
		</tr>
		<tr>
			<td>PlusOne Repel Silane</td>
			<td>GE Healthcare Biosciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-digoxigenin AB</td>
			<td>Roche applied science</td>
			<td>11093274910</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 4: Preparation of seeds and segmented microtubules.</td>
		</tr>
		<tr>
			<td>Tubulin</td>
			<td>purified from cow brains 
			Cytoskeleton, Inc</td>
			<td>&#160; 
			T238P</td>
			<td>For purification protocols see 49&#8211;51 
			Unlabeled porcine tubulin</td>
		</tr>
		<tr>
			<td>Labeled tubulin</td>
			<td>Cytoskeleton, Inc 
			Invitrogen 
			Invitrogen</td>
			<td>TL590M 
			C1171 (Rhodamine) 
			A-2952 (Digoxigenin)</td>
			<td>Rhodamine-labeled porcine tubulin 
			Tubulin can be labeled with any amine-reactive dye as in reference52.</td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jena Biosciences</td>
			<td>NU-405</td>
			<td>Aliquot and store at -70 &#176;C</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td>vaseline, lanolin, and paraffin at 1:1:2 by mass</td>
			<td></td>
			<td>see ref. 9</td>
		</tr>
	</tbody>
</table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

미세 소관 해중합은 체외에서 다른 단백질 복합체 단백질 코팅 비즈를 수송하는 힘을 제공 할 수 있습니다. 기본 메커니즘은 세포 분열 중에 미세 소관 의존 염색체 운동에 중요한 역할을하는 것으로 생각되지만, 관련 단백질과 자신의 정확한 역할은 불분명합니다. 따라서, 정제 된 구성 요소와 정의 생화학 적 환경을 사용하여 체외에서 이러한 운동을 연구하는있는 분석법을 개발하는 요구가 증가하고 있습니다. 미세 소관은, 그러나, 본질적으로 불안정 중합체이다, 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 우리가 여기에서 설명하는 프로토콜은 photoablatable는 캡을 안정으로 만든 분할 된 미세 소관을 활용. 이러한 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝에서 운동의 연구를 지원, 높은 공간적 해상도로 트리거 될 수 있습니다. 이 기술은 CA에 사용될 수있다동적 미세 소관은 끝 processively 이동 형광 표지 된 단백질 복합체, 분자의 수의 정량적 인 분석을 RRY. 이것으로 신호 대 잡음 비율 및 기타 정량적 형광 분석법을 최적화하려면, 커버 슬립은 수용성 형광 표지 된 단백질의 비특이적 흡착을 감소하도록 처리한다. 상세한 프로토콜은 고려 형광 조명의 불균일을 취해, 등거리 가우시안 적합하여 하나의 형광의 강도를 결정하기 위해 제공된다. 마지막으로, 우리는 processive화물 운동에 몇 미세 소관 해중합에 다른 모터와 nonmotor 단백질의 기능에 대한 통찰력을 제공하는 단백질 코팅 마이크로 비즈의 미세 소관에 의존하는 동작을 연구하는 분할 된 미세 소관의 사용을 설명합니다.

미세 소관을 해중합과 추적 단백질이 끝납니다. 효모 동원체 구성 요소 Dam1는 지금까지 해중합 미세 소관의 최고의 팁 트래커 (14)를 종료한다. GFP로 ​​표지이 10-서브 유닛의 복잡한 쉽게 표현 박테리아 세포 18,38에서 정제, 그래서 우리는 끝 추적 분석에 대한 긍정적 인 제어로 사용하는 것을 권장 할 수 있습니다. 미세 소관의 해중합 끝으로 추적 형광 단백질은 꾸?...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

미세 소관은 본질적으로 불안정 폴리머, 그리고 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 여기에서 우리는 photoablatable 안정 캡으로 분할 미세 소관을 사용하여 프로토콜을 설명합니다. 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝 동작의 분석을 지원, 높은 공간적 해상도로 트리거 될 수 있습니다.

분해 미세 소관 엔드에 힘 입어 모션을 연구하기 위해 분단 미세 소관의 준비

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Research

JoVE Journal

Biology

14.3K 조회수.  Russian Academy of Sciences.  미세 소관은 본질적으로 불안정 폴리머, 그리고 성장과 단축 사이에 자신의 전환 확률 통제하기가 어렵습니다. 여기에서 우리는 photoablatable 안정 캡으로 분할 미세 소관을 사용하여 프로토콜을 설명합니다. 분할 된 미세 소관 해중합함으로써 분해 미세 소관의 끝 동작의 분석을 지원, 높은 공간적 해상도로 트리거 될 수 있습니다. 

비디오: Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

의 분리 및 정제 Drosophila</em마그네틱 비드별로 정렬> 주변 뉴런

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

생산<em&gt; Xenopus의 tropicalis</em&gt; 달걀은 미세 소관 관련 RNA를 식별하기 위해 추출

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

MBP 기반 태깅 전략에 의해 Mgm101 재조합 단백질의 제조

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

소장에 의해 지질 전송을 연구하기 위해 카코 2 셀을 사용하여

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

단극성 미토틱 스핀들에서 디스크 현미경 검사법에 의한 미세소관 역학 측정

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

에슈리치아 대장균에서 모토렐라 구동 운동성 조사

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

모기 전기 조영술에 대한 단계별 가이드

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

루트 엑수데이트에서 뿌리 균 화학 물질의 신속 한 식별에 대 한 향상 된 화학 분석

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...