Name: visualizing clathrin-mediated endocytosis of g protein-coupled receptors at single-event resolution via tirf microscopy
Uploaded: 2014-10-20T13:00:00
Description: Watch this Scientific Journal Video about Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy at JoVE.com

The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.

찬란의 1 발현은 배양 된 세포에 CME 구성 요소 태그 HEK293 세포는 GPCR 생물학 및 세포 내 이입을 연구하기 위해 광범위하게 사용되고있다, 따라서이 프로토콜의 모델로 사용되는 유용한 모델 세포입니다. 과발현과 낮은 세포 독성이없는 균일 한 표현을 제공하는 형질 전환 프로토콜을 사용합니다. <ol><li> 멸균 층류 후드에있는 모든 세포 배양을 처리합니다. 10 % 소 태아 혈청 ...

<ol>
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여기에서 우리는 실시간으로 살아있는 세포에서 개별 CCPS의 수준에서 clathrin 매개 엔도 시토 시스 (CME)를 시각화하는 TIRFM의 사용을 설명합니다. CME는 많은 공간적으로 시간적으로 별도의 개별 이벤트의 누적 효과에 의해 매개 신속하고 매우 동적 인 이벤트입니다. 이러한 내재화의 생화학 적 측정과 같은 현재 사용되는 대부분의 분석법, 표면 바이오 티 닐화 또는 리간드 결합, 유동 세포 계측법 ?...

clathrin 매개 엔도 시토 시스 (CME)의 과정은 소체 1-3에화물을 수집하고 세포막을 조작 clathrin-매개 된 세포 내 이입 기계의 다양한 컴포넌트뿐만 도착 타이밍에 의존한다. CME는 엔도 시토 시스 1의 초기 사이트에서 함께 와서 막 변형 및화물 어댑터 단백질에 의해 시작된다. 이러한 단백질은 clathrin 코팅 피트 (CCP) (4)을 형성 새장 형 구조로 어셈블 외피 단백질 clathrin를 모집. CCP가 완전히 주로 큰 GTPase, dynamin의 작용을 통해 구형, 막 분열로 조립되면, 무료 clathrin 코팅 소포 (CCVS) 5,6를 생성합니다. 이러한 내재화는 구성 요소의 여러 CME 라운드 동안 재사용 될 수 있도록 clathrin 코트의 신속한 분해를 트리거한다. CME에 관여하는 단백질의 발견 및 특성화는 전통적인 생화학 뿌리되었습니다emical, 유전 및 현미경 기술 4-6,8. 이러한 분석법은 이러한 세포 내 이입 컴포넌트 역할과 상호 작용 지점을 해명 하였다. 인신 매매 기계의 필수 구성 요소를 정의하기위한 매우 유용하지만, 이러한 분석은 매우 CME 구성 요소 나화물 농도의 동적 동작을 캡처 제한됩니다. CME 정의 단계에서 단백질의 모듈 세트의 안무 조립체에 의해 구동되기 때문에 이것은 중요한 제한이 있으며, 개개의 세포 내 이입 이벤트 역학의 작은 변화는 세포 내 이입에 큰 누적 결과를 초래할 수 있기 때문이다. 또한, 최근의 데이터는 개별 CCPS이 과정의 생리적 조절이 높은 공간과 시간적으로 9-14 제약을 시사 조성과 행동 모두 다를 수 있음을 나타냅니다. 각각의 세포 내 이입 이벤트를 시각화 따라서, CME에 관련된 여러 중복 단백질과 방법이 단백질을 제어 할 수 있습니다 B가 이유를 이해하는 것이 필수적입니다Y 생리 학적 신호는화물 국제화를 조절합니다. 여기에서 우리는 살아있는 세포의 개별 CCPS의 역학의 수준에서 CME를 관찰하는 총 내부 반사 형광 현미경 (TIRFM)의 사용을 설명합니다. TIRFM는 유리 커버 슬립 셀 (15, 16)의 유체 환경 사이의 굴절율의 차이에 의존한다. 여기 광이 임계각 이상에서 세포쪽으로 이동 될 때, 내부적으로는 커버 슬립 위에 약 100 nm의 연장 조명 얇은 필드를 유지 소멸 파를 생성 반사된다. 이것은이 좁은 필드 내에서만 형광 분자가 흥분 보장합니다. 실제적으로, 이것은 나 세포막 근처 형광 물질의 여기를 허용하고, 셀의 내부 부분으로부터 형광을 최소화한다. 이것은 세포막 공동에서 이벤트를 시각화하는 상당히 높은 신호 대 잡음비 및 z 축 해상도를 제공종래의 표면 형광 또는 공 초점 형광 현미경으로 더 일반적으로 사용되는 모드로 mpared. 우리는 또한 입문 실용 레벨에서 설명하는, 일반적으로 사용되는 이미지 분석 플랫폼의 사용을 분석하고 간단한 형태 학적 특징 및 개별화물 세포 내 이입 이벤트 역학을 정량한다.

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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:207px;">Comments/Description</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">DMEM/High Glucose with L-glutamine and sodium pyruvate</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>SH3024301</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS), no calcium, no magnesium</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21600-010</td>
			<td></td>
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			<td height="40" style="height:40px;width:367px;">EDTA Free Acid</td>
			<td style="width:180px;">Amresco</td>
			<td style="width:157px;">0322-500G</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Fetal Bovine Serum</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;">10437-028</td>
			<td></td>
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			<td height="40" style="height:40px;width:367px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td>21083-027</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Opti-MEM</td>
			<td style="width:180px;">Gibco, by Life Technologies</td>
			<td style="width:157px;"></td>
			<td></td>
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			<td height="40" style="height:40px;width:367px;">HEPES</td>
			<td style="width:180px;">CellPURE by Fisher Scientific&#160;</td>
			<td>BP2937-100</td>
			<td></td>
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			<td height="40" style="height:40px;width:367px;">Effectene&#160;</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:157px;">301425</td>
			<td>Transfection reagent</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:367px;">25 millimeter coverglass</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>12-545-86&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Corning cell culture treated flasks, 25cm2</td>
			<td style="width:180px;">Fisher Scientific&#160;</td>
			<td>10-126-28</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Cell culture 6-well plate</td>
			<td style="width:180px;">Greiner Bio-One, by VWR</td>
			<td>82050-896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Monoclonal ANTI-FLAG M1</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>F3040-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">[D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO)</td>
			<td style="width:180px;">Sigma Aldrich</td>
			<td>E7384-5MG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Alexa Fluor 647 Protein Labeling Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:157px;">A20173</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">ImageJ</td>
			<td style="width:180px;">NIH</td>
			<td style="width:157px;"></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Imaris Image analysis software</td>
			<td style="width:180px;">BitPLane</td>
			<td style="width:157px;"></td>
			<td>http://www.bitplane.com/imaris/imaris, for automated analysis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Nikon TIRF arm with required adapters for Nikon Eclipse Ti</td>
			<td style="width:180px;">Nikon</td>
			<td style="width:157px;"></td>
			<td>For adjusting angle of incidence</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">iXon+ EMCCD camera and adapters</td>
			<td style="width:180px;">Andor</td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing clathrin-mediated endocytosis of g protein-coupled receptors at single-event resolution via tirf microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

사용 라이브 셀 TIRF 현미경 우리는 μ-오피오이드 수용체 (MOR)의 세포 내 이입 역학을 녹음 한, G-단백질 결합 수용체 (GPCR)와 세포 내 이입 어댑터 단백질 β-arrestin. β-arrestin 구조체는 일시적으로도 1에 설명 된 프로토콜을 사용하여 안정하게 발현하는 MOR 세포주에 형질 전환하고, 96 시간 후 묘화 하였다. 안정적인 세포주의 MOR은 N-말단의 pH에​​ 민감한 GFP 태그입니다. 이 형광 단백질?...

Watch this Scientific Journal Video about Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy at JoVE.com

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF 현미경을 통해 단일 ​​이벤트 해상도에서 G 단백질 결합 수용체의 Clathrin 매개 엔도 시토 시스를 시각화

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Research

JoVE Journal

Biology

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

TIRF 현미경과 망막 바이폴라 세포 이미징 Exocytosis

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

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생물학

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

이미징 G - 단백질 결합 수용체 (GPCR) - 중재 신호 행사 그 제어 Chemotaxis Dictyostelium Discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

G 단백질 결합 수용체에 작용제 활동 Quantifying

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

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형광 현미경을 사용하여 Nematodes에 박테리아를 머릿속으로

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

유전자 인코딩 분자 프로브는 G 단백질 결합 수용체를 연구하는

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

고속 3D 구조 조명 현미경을 사용하여 라이브 박테리아의 Cytokinetic Z 반지의 슈퍼 해상도 이미징 (F3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

형광 기반의 칼슘 동원 분석에 의해 G 단백질 결합 수용체의 특성

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

TIRF 현미경을 통해 단일 ​​이벤트 해상도에서 G 단백질 결합 수용체의 Clathrin 매개 엔도 시토 시스를 시각화

TIRF 현미경을 통해 단일 이벤트 해상도에서 G 단백질 결합 수용체의 Clathrin 매개 엔도 시토 시스를 시각화