저자 감사 Benoit Sorre, Damien Cuvelier, 피에르 Nassoy, 프랑수아 Quemeneur, 및 그들의 필수 기여에 대 한 길 Toombes 그룹에서 나노튜브 메서드를 설정 합니다. 샌드위치 그룹 속하는 CNRS 컨소시엄 CellTiss, Labex CelTisPhyBio (ANR-11-LABX0038), 그리고 파리 과학 외 Lettres (ANR-10-IDEX-0001-02). F. C. Tsai EMBO 장기 친교 (ALTF 1527-2014)과 마리 퀴리 행동 (H2020-MSCA-IF-2014, 프로젝트 막 ezrin 말라)에 의해 투자 되었다. 석사 시 몬스 사회 휄 로우의 주니어 연구원 이다.

저자는 공개 없다.

하지만, 막의 기본적인 기계적 특성 측정을 의미 하지만 단백질 및 막 간의 결합에 빛을 발산 하는 데 도움이 막 단백질 시스템에 풍부한 정보를 제공 하는 GUVs에서 튜브를 당기기의 방법 곡률입니다. 소개에서 설명 했 듯이, 다른 기술을 서브 미크론 리19 세 표면18,곁에와 단백질을 배양 하거나 관찰 하 여 막 커브 단백질의 효과 측정 하기 위해 존재 합?...

Endocytosis, 인신 매매, filopodia, 감염 등의 형성 등 많은 세포질 과정 세포 막1,2의 모양에 있는 극적인 변화를 함께 제공 됩니다. 셀에 다양 한 단백질 막에 바인딩 및 그들의 모양을 변경 하 여 이러한 프로세스에 참여 하 고. 가장 주목할 만한 보기는 본질적으로 도메인3,,45,,67바 곡선 특성을 포함 하는 빈/Amphiphysin/Rvs (바) 단백질 가족의 일원. 일반적으로, 그들은 표면에 그리고, 많은 경우에, 또한 얕게 삽입은 bilayer amphipathic 나선 바 도메인 준수는 막으로 작용. 모양, 크기, 및 amphipathic 나선 수와 함께 바 도메인의 결정: (1) 막 곡률의 방향 (즉, 여부 그들은 invaginations 또는 돌출을 유발 한다), 및 (2) 막의 크기 곡률의5,8. 노트, 여기 긍정적인 곡률 달리 곡선된 막, 즉, 상호 작용 입자, 그리고 부정적인 돌출의 볼록한 쪽으로 정의 됩니다. 또한, 단백질 바의 양적 연구는 막에 그들의 효과 물리적 매개 변수 집합에 따라 달라 집니다 공개: 표면 단백질, 막 긴장, 그리고 막 모양 (평면 대 관 대 구형의 밀도 모양)7. 단백질 바 이러한 매개 변수에 따라 수 있습니다: (1) 막 곡률의 센서 역할 (2) 구 부 막, 또는 (3) 유도 막 절단7.
막 셀에 재편 endocytosis, 같은 현상의 양적 측면을 공부에 관련 된 부품의 투명 한 수 비보에 매우 도전 이다. 최소한의 구성 요소를 셀에 곡선된 막을 흉내 낸의 생체 외에서 재구성 어떻게 막 커브 단백질의 작동 기계 이해를 얻기 위하여 수단을 제공 합니다. 이 문서는 막 나노튜브에서 생체 외에서 micromanipulation, confocal 현미경 검사 법, 광학 핀셋을 사용 하 여 다시 구성할 프로토콜을 설명 합니다. 접근은 공부 하 고, 양적 방법, 단백질, 지질, 또는 작은 분자 곡선된 막 상호 작용 하는 방법을 사용할 수 있습니다. GUVs 지질 누구의 곡률 막 커브 상호 작용 분자의 크기에 비해 무시할 수은 세포 막의 모델로 사용 됩니다. 그들은 electroformation 방법9 는 소포 지질 영화를 수 화와 교류 전류 (AC)10에서 GUVs로 붓기 형성 된다를 사용 하 여 준비 된다. 가장 일반적인 기판 GUVs 재배 되는 인듐 주석 산화물 (ITO) 코팅 중 반 전도성 격판덮개 또는 백 금 철사 (Pt-전선)11. 이 작품에서는, GUVs는이 방법은 버퍼12소금의 면 전에 서 GUVs를 만드는 대안 보다 훨씬 더 잘 작동 하도록 표시 되었습니다로 Pt-전선에 재배 됩니다. Electroformation 프로토콜은 여기에 설명 된 그것을 재현 하는 충분 한 세부 사항에, 하지만 우리는 비슷한 다른 메서드와 GUVs를 만드는 세부 사항13,14에서 설명 되었습니다 이전 기사에 독자를 참조 하십시오. 우리 손에 Pt 전선에 electroformation는 성공적으로 나왔고 GUVs 또는 자연 지질 합성 지질의 혼합에서 ~ 100 m m NaCl을 포함 하는 버퍼에 추출 물. 또한, 성장 하는 동안 GUVs 내부 단백질을 캡슐화 가능 했다 또한. 예를 들어 electroformation 챔버는 그림 1A;에 나와 그것은 2 ~ 10 cm 긴 Pt-와이어 홀더 유리 coverslips와 양쪽에 밀봉 될 수 있다 소계 (PTFE)에서 만든 삽입 구성 ~ 1-2 c m 간격 (그림 1A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56086/56086fig1.jpg"> 그림 1: 실험적인 체제. Pt-와이어에 연결 된 전기 커넥터 (A) 우두머리 electroformation 챔버. (B) 왼쪽: 현미경을 보여주는 실험 시스템, 목표 및 2 개의 micropipettes (왼쪽 및 오른쪽) 위 실험 챔버는 micromanipulators에 연결 된 고 튜브 고 단백질에 대 한 실험 챔버에 삽입 사출입니다. 오른쪽: 실험 챔버의 근접 보기는 포부와 삽입 분사 micropipettes의 끝을 보여주는 목표 위에 탑재. (C) 주사기의 백 엔드에 micropipette 삽입 얇은 디스펜서 장비. 하단의 파란색 점선 개요는 micropipette micropipette 내부 디스펜서의 근접 보기입니다. 이 시스템은 카 세 인 유리 표면 passivate 하 고 필요할 때 미네랄 오일 칠을 다시 하는 micropipette 채우기 위해 사용 됩니다. (D) A 시스템 µ L 양의 단백질 해결책을 발음 하는 데 사용 합니다. 바늘은 주사기와 주입 micropipette에 연결 된 튜브에 연결 되어 있습니다. Micropipette 팁 신중 하 게 단백질 해결책에 몰입 하 고 그렇게 발음 하는 micropipette 팁을 채우기 위해. micropipette 다음 다시 채워집니다 미네랄 오일 패널 C.에에서 표시 하는 시스템을 사용 하 여 <a href="//cloudflare.jove.com/files/ftp_upload/56086/56086fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
막 나노튜브, 7에서 반경에 이르기까지 몇 백 nm nm는 우두머리에서 외부 힘으로 당길 수 있다. 이 방법은 처음 세포 막 및 소포, 휨 강성15,16등의 탄성 속성 측정 하도록 설계 되었습니다. 가장 최근 작품, 메서드 가져온된 나노튜브7,17근처 단백질 microinjecting 곡선된 멤브레인과 단백질의 상호 작용 연구를 확대 되었다. 다른 방법 막 커브 단백질 연구 개발 되었습니다. 한 방법에서는, 단백질 세 표면에 닿는 곳에 다르게 크기의 리와 알을 품는. Confocal 현미경 검사 법은 단백질 바인딩 곡률을 이용한 정렬18,19를 나타낼 수 있는 liposome 직경의 함수로 측정 하는 데 사용 됩니다. 다른 방법에서는, 단백질 tubules20,21을 자발적으로 유도 하는 능력을 측정 하기 위해 마이크로 발음 하는 우두머리 근처 주입 됩니다. 이 프로토콜에서 설명 하는 방법을 막 커브 단백질 endocytosis에 관련 된 공부에 특히 적합 한은 대부분 단백질이 일반적으로 미리 형성한 막 나노튜브와 함께 화물 포함 된 막 invagination 연결을 발생 합니다 평면 플라즈마 멤브레인 기본 또한,이 방법에서는, 달리 곁된 작은 리와 분석 결과에 막 나노튜브는 지속적으로에 연결 막; 따라서, 그것은 한 상황으로 예상 vivo에서우두머리와 기계적 평형 에입니다. 따라서, 근본적인 막 물리학 적용 하 고 우리가 우리의 측정22,,2324에서 기계적 특성의 과다를 추정할 수 있다.
이 방법의 전체 구현에 대 한 필요한 장비는 confocal 현미경, 광학 핀셋, 그리고 하나 또는 두 개의 micropipettes 물 탱크 (그림 1B)에 연결을 포함 합니다. 모든 3 개를 결합해 서, 동시에 막 긴장, 막 곡률, 단백질의 표면 밀도 측정 하 고 힘25를 튜브에 가능 하다. Micropipette 포부 필수적 이며, 그것은 쉽게 액체 정역학 압력을 통해26포부 압력 제어 하는 물 탱크에 연결 홀더 유리 제 micropipette 삽입 하 여 구성 됩니다. micropipette 홀더는 제어는 micromanipulator 고, 이상적으로, 한 방향으로 정밀 운동 위한 압 전 액추에이터. 나노튜브를, microaspirated 우두머리는 짧게 미크론 크기의 구슬을 다음 멀리는 나노튜브를 만드는에 붙어 됩니다. 이 구현에는 구슬27게시 된 프로토콜 따라 생성 될 수 있다 광학 족집게에 의해 개최 됩니다. 그것이 비록 정확한 힘 측정 비용 다른 방법으로, 광학 족집게 및 풀 나노튜브의 분배 가능 합니다. 광학 트랩을 만들려고 너무 어려운 경우 또는 힘 측정은 중요 하지,와 같은 경우 단순히 곡선된 멤브레인에 대 한 단백질의 선호를 확인 하 고 싶어 하는 경우 튜브 발음 하는 두 번째 micropipette28의 끝에 비드를 사용 하 여 가져온 수 있습니다. 그것은 또한30,31흐름 또는 중력29 를 사용 하 여 튜브를 당겨 수 있습니다. 또한, confocal 현미경 검사 법 중요 하지 않습니다. 그러나, 그것은 단백질의 표면 밀도 측정 하기 위해 그렇게 선호. 그것은 또한 지질 막 힘 및 긴장은 별도로 따라서 튜브의 형광 강도에서 나노튜브 반경 측정 있습니다. 형광에서 추론 튜브 반경 이러한 수량 사이의 관계 막 준수 단백질25의 존재로 인해 잘 설립 방정식에서 일탈 하는 경우에 특히 중요 하다. 중요 한 것은, 하나로 튜브 곡률을 측정할 수 있을 것입니다 광학 트랩 confocal 현미경 검사 법의 분배 수 없습니다.
이 프로토콜에서 설명 된 대로 메서드 나노튜브는 바 가족25,,3233,34에서에서 주로 그들에 다양 한 주변 막 단백질의 곡률을 이용한 정렬 공부 하는 데 사용 되었습니다. . 그것은 또한 KvAP에 농축 conically 모양의 막 횡단 칼륨 채널 단백질35바 같은 방식으로 나노튜브 곡선을 표시 했다. GUVs 내부 단백질을 캡슐화 하는 메서드를 최적화 하 여 부정적인 곡률과 단백질의 상호 작용은 최근 조사 잘36로. 또한,이 메서드는 사용 단백질 건설 기계25,37 의 형성을 명료 하 고 중 선 긴장38단백질 dynamin39, 또는 바 막 절단의 메커니즘을 연구 하 단백질40,41. 뿐만 아니라 단백질, 작은 분자 또는 이온 곡률을 유도할 수 있다 또한. 이 메서드를 사용 하 여 칼슘 이온 소금 없는 조건42에서 긍정적인 곡률을 유도 하기 위해 표시 했다. 흥미롭게도, 그것은 또한 보였다 지질 demixing 포인트43,44근처는 작곡에만 있지만 정렬, 곡률을 받을 수 있습니다. 합계에서는, 연구원은 어떻게 중 정수 막 구성 요소 (예:, 지질 또는 막 횡단 단백질) 조사에 관심이 메서드를 사용할 수 있습니다 또는 주변 분자 바인딩 (중 내부 또는 외부 GUVs)와 상호 작용 기계 및 양적 관점에서 원통 곡선된 막 그것은 또한22,,2345막 자체의 기계적 성질 측정에 관심 있는 사람을 위한 것입니다.

<table>
	<tbody>
		<tr>
			<td>1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)</td>
			<td>Avanti</td>
			<td>850375</td>
			<td>Example lipid used in data for Figure 3</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] [DSPE-PEG(2000)-biotin]</td>
			<td>Avanti</td>
			<td>880129</td>
			<td>biotinylated lipid</td>
		</tr>
		<tr>
			<td>BODIPY-TR-C5-ceramide</td>
			<td>Molecular Probes (ThermoFisher)</td>
			<td>D7540</td>
			<td>fluorescent lipid</td>
		</tr>
		<tr>
			<td>BODIPY- FLC5-hexadecanoyl phosphatidylcholine (HPC*)</td>
			<td>Molecular Probes (ThermoFisher)</td>
			<td></td>
			<td>fluorescent lipid for protein density calibration</td>
		</tr>
		<tr>
			<td>egg L-&#945;-phosphatidylcholine (eggPC)</td>
			<td>Avanti</td>
			<td>840051</td>
			<td>used for calibrating the tube radius constant</td>
		</tr>
		<tr>
			<td>&#946;-casein from bovine milk (&#62;99%)</td>
			<td>Sigma Aldrich</td>
			<td>C6905</td>
			<td>used for passivating the micropipette and the experimental chamber</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+) glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma Aldrich</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>osmometer</td>
			<td>Loser</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Pt-wires, 0.5 mm diameter, 99.99% pure</td>
			<td>Goodfellow USA</td>
			<td>PT005139</td>
			<td>used for GUV electroformation</td>
		</tr>
		<tr>
			<td>function generator</td>
			<td></td>
			<td>n/a</td>
			<td>used to create current for GUV electroformation</td>
		</tr>
		<tr>
			<td>putty sealant</td>
			<td>Vitrex (from CML France)</td>
			<td>CRIT 140013</td>
			<td>used to seal the electroformation chamber</td>
		</tr>
		<tr>
			<td>bath sonicator</td>
			<td></td>
			<td>n/a</td>
			<td>useful to clean the electroformation chamber, but not crucial</td>
		</tr>
		<tr>
			<td>Nikon TE2000 inverted microscope, eC1 confocal system (Nikon), with two laser lines (&#955; = 488 nm and 543 nm); optical tweezers induced by a 5 W ytterbium fiber continuous wave laser (&#955; &#62; 1070 nm; IPG GmBH Germany)</td>
			<td></td>
			<td></td>
			<td>an example of a confocal microscopy system equipped with optical tweezers</td>
		</tr>
		<tr>
			<td>micromanipulators</td>
			<td></td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate capillaries (with internal and external radii of 0.78 mm and 1 mm, respectively)</td>
			<td>Harvard apparatus</td>
			<td>30-0036</td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>microforge</td>
			<td>Narishige</td>
			<td>MF-800</td>
			<td></td>
		</tr>
		<tr>
			<td>piezoelectric actuator</td>
			<td>Physik Instrumente</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene streptavidin coated beads (diameter 3.2 &#181;m)</td>
			<td>Spherotech</td>
			<td>SVP-30-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

pulling membrane nanotubes from giant unilamellar vesicles

세포 막의 재편 endocytosis, 인신 매매, 등 filopodia형성 등 많은 세포 현상의 중요 한 부분입니다. 많은 다른 단백질 그들의 능력을 감지 하거나 유도 막 곡률 때문에 곡선된 멤브레인과 연결 합니다. 일반적으로 이러한 프로세스는 다양 한 단백질 셀에서 양이 많게 공부 하기에 너무 복잡 하 포함 한다. 우리는 endocytic 목 같은 곡선된 세포 구조를 흉내 낸는 곡선된 막에 생체 외에서다시 구성할 프로토콜을 설명 합니다. 거 대 한 unilamellar 소포 (우두머리)는 그의 내부 압력 및 표면 장력 micropipette 포부와 함께 제어 되는 세포 막의 모델로 사용 됩니다. 광학 핀셋을 사용 하 여 우두머리에는 포인트 당기는 힘을 적용 평면 막에 연결 된 높은 곡률의 나노튜브를 만듭니다. 이 방법은 전통적으로 굽 힘 강성 등 지질 막의 기본적인 기계적 특성을 측정 하기 위해 사용 되었습니다. 최근 몇 년 동안, 그것은 단백질 막 곡률 및 그들은 모양과 막의 역학에 영향을 미칠 방법으로 상호 작용 하는 방법을 공부 하 확장 되었습니다. Micromanipulation, microinjection, 광학 핀셋, confocal 현미경 검사 법을 결합 한 시스템 동시에 막 곡률, 막 긴장, 그리고 단백질의 표면 밀도의 측정을 허용 한다. 이 측정에서 단백질 막 시스템의 많은 중요 한 기계 및 형태학 속성 유추 수 있습니다. 또한, 우리 하다 생리 소금 농도 형광 강렬 레이블이 단백질과 지질에서에서 막에 단백질의 표면 밀도 측정 하는 방법의 GUVs를 만드는 프로토콜.

튜브를 당기 실험 멤브레인에 대 한 중요 한 기계적인 정보를 줄 수 있습니다. 단백질 또는 다른 분자를 막 곡률, 막와 커플 하 고 튜브 반경 막 긴장 Canham Helfrich 해밀턴을 적용 하 여 관련 될 수 없을 경우에는 튜브를 방정식 우두머리50,51에서 가져온 
<img alt="Equation 3" src="/files/ftp_uplo...

Watch this Scientific Journal Video about Pulling Membrane Nanotubes from Giant Unilamellar Vesicles at JoVE.com

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

셀에 많은 단백질을 감지 하 고 막 곡률을 유도. 곡선된 막 체 외단백질 또는 어떤 곡률 활성 분자의 상호작용을 연구 하는 지질 소포에서 막 나노튜브를 당겨 하는 방법을 설명 합니다.

거 대 한 Unilamellar 소포에서 막 나노튜브를 당기

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

pulling-membrane-nanotubes-from-giant-unilamellar-vesicles

Research

JoVE Journal

Biology

10.9K 조회수.  PSL Research University, CNRS UMR168. 셀에 많은 단백질을 감지 하 고 막 곡률을 유도. 곡선된 막 체 외단백질 또는 어떤 곡률 활성 분자의 상호작용을 연구 하는 지질 소포에서 막 나노튜브를 당겨 하는 방법을 설명 합니다.

비디오: Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

연구

생물학

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

대형 Unilamellar Vesicles로 펩타이드의 Translocation 측정

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

대형 Unilamellar 소포와 검사 BCL-2 가족 기능

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

이미징 및 패치 클램프 전기 생리학에 대한 거대한 리포좀 준비

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

횡단 단백질의 재구성, 현미경 및 패치 클램프 연구 거대한 단일 층 소포에 전압 게이트 이온 채널, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion 기술은 쥐 장간막에서 미세 혈관 투과성의 규정을 조사하기 위해

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

포유류 세포에 있는 바닥 상태 고갈 슈퍼 해상도 영상

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Extracellular 소포 세포 조절 미디어에서의 Cytometric 분석 흐름

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

대장균 및 다른 박테리아로부터의 세포외 소포의 확장 가능한 분리 및 정제

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...