이 작품은 건강의 국가 학회 [R01GM121847]에 의해 지원되었다; 기념 슬론 케터링 암 센터 (MSKCC) 지원 보조금 / 핵심 보조금 (P30 CA008748); MSKCC 기능 유전체학 이니셔티브.

저자는 공개 할 것이 없습니다.

전형적인 시험관내 TIRF 단일 분자 실험과 비교하여, 여기에 기재된 분석기를 사용한 단일 분자 이미징은 세포 추출물의 사용과 형광으로 표기된 항체의 고농도로 인해 더욱 복잡하다. 표면 PEGylation의 한 라운드의 일반적인 관행에 비해, PEGylation (단계 2)의 두 번째 라운드는 검출 표면(15)에비특이적 항체 결합을 크게 감소시킨다. 확산형광 항체의 고농도는 항체 결합의 단?...

진핵 계통에서의 번역은 주로 7-메틸구아노신(m7G) 캡 의존경로1을통해 발생한다. 연구에 따르면 진핵 번역의 개시 단계는 속도 제한및 규정2,3,4에대한 공통의 목표임을 나타낸다. 캡 의존형 번역의 메커니즘은유전5,생화학6,7,8,구조9,및 게놈10 벌크 접근법을 사용하여 광범위하게 연구되었다. 이러한 방법은 캡 의존개시를 조절하는 다양한 메커니즘을 확인했지만, 해상도는 이기종 및 비동기 개시 이벤트에서 신호의 평균을 앙상블로 제한합니다. 최근에는 생체내 번역 이벤트가 초기 폴리펩티드(11,12,13,14)에대한에피토프에 대한 형광 항체 결합을 측정하는 방법에 의해 시각화되었다. 그러나, 이러한 새로운 접근법은 또한 다중 형광 항체가 높은 세포내 형광 배경에서 단일 번역 이벤트를 해결할 수 있도록 초기 펩티드를 결합해야 하기 때문에 개별 개시 사건을 해결하는 능력에 제한됩니다. 많은 생물학적 상호 작용에서, 해결된 개별 운동 사건은 분자 수준에서 동기화할 수 없는 복잡한 다단계 및 반복적인 생물학 프로세스를 이해하는 중요한 통찰력을 제공했습니다. 캡 의존개시 및 규제를 더 잘 이해하기 위해서는 개별 번역 이벤트의 역학을 추적할 수 있는 새로운 방법이 필요합니다.
우리는 최근에 단 하나 분자 분해능15를가진 캡 의존성 개시 운동학을 측정하는 체외 분석서를 개발했습니다. 이 개시 경로3,16에관여하는 많은 공지되고 알려지지 않은 단백질 인자를 고려하여, 단일 분자 분석체는 세포인자 및 견고한 번역활동(17,18,19,20,21,22,23,24, 25,25)의보존으로부터 이익을 얻는 기존의 체외 세포 없는 번역 시스템과 호환될 수 있도록 개발되었다. 또한, 세포 없는 번역 시스템의 사용은 단일 분자 관측및 이전 대량 결과 사이 더 호환 비교를 허용합니다. 이 접근법은 캡 의존개시의 기존 기계론적 프레임워크에 새로운 단일 분자 운동 통찰력의 직진 통합을 제공합니다. 단일 분자 분석방식을 확립하기 위해, 기존의 세포없는 번역 시스템은 세 가지 방법으로 변형된다: 에피토프 인코딩 서열은 리포터 mRNA의 개방 독서 프레임 (ORF)의 시작 부분에 삽입된다; 기자 mRNA의 3′ 끝은 단일 분자 검출 표면에 mRNA 최종 테더링을 용이하게하기 위해 바이오티닐화된다; 형광으로 표지된 항체는 번역 추출물로 보충된다. 이러한 수정은 기본적인 분자 생물학 기술과 일반적으로 사용할 수있는 시약만 필요합니다. 더욱이, 이러한 변형 및 단일 분자 이미징 조건은 대량 세포 없는 번역 반응의 번역 역학을보존한다(15).
이 분석체(그림1)에서5′-end 캡 및 3′-end 바이오타이니드 리포터 mRNA는 유동 챔버에서 스트렙타비딘 코팅 검출 표면에 고정된다. 유동 챔버는 형광으로 표지된 항체로 보충된 세포없는 번역 혼합물로 채워져 있습니다. mRNA 번역후 약 30-40코돈 하류의 에피토프서열(26,27)을위해 발생한 후, 에피토프는 리보솜 출구 터널에서 나오고 형광으로 표지된 항체와 상호 작용할 수 있게 된다. 이러한 상호 작용은 급속하고 단일 분자 형광 이미징 기술에 의한 검출을 통해 활성 세포 없는 번역 중에 단일 분자 분해능으로 번역 역학을 추적할 수 있습니다. 이 분석은 캡 의존성 번역 역학 및 규제, 특히 체외 분석에서 대량 으로 작동하는 시스템의 경우 체외 연구에서 광범위하게 이점을 얻을 수 있습니다.
이러한 단일 분자 분석법을 확립하기 위한 전제 조건은 이전에 설명된방법(28)에따라 상업적으로 사용 가능하거나 제조된 번역 추출물을 사용하여 달성될 수 있는 대량 세포 없는 번역 분석이다. 진핵 번역 추출물은 곰팡이, 포유류 및식물(28)을포함한 다양한 세포로부터 얻을 수 있다. 이미징의 경우, 이 분석법은 튜닝 가능한 레이저 강도 및 사고 각도, 전동 식 샘플 단계, 동력 유체 시스템 및 샘플 온도 제어 장치가 장착된 TIRF 현미경이 필요합니다. 이러한 요구 사항은 현대 체외 단일 분자 TIRF 실험에 대한 일반적이며 다르게 달성 될 수있다. 여기에 제시된 실험은 재료표에나열된 시판되는 현미경, 소프트웨어 및 액세서리로 구성된 객관적인 유형의 TIRF 시스템을 사용합니다.

<table>
	<tbody>
		<tr>
			<td>100X oil objective, N.A. 1.49</td>
			<td>Olympus</td>
			<td>UAPON 100XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/bis (40%, 19:1)</td>
			<td>Bio-Rad</td>
			<td>161-0144</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline liquid detergent</td>
			<td>Decon</td>
			<td>5332</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane (N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane)</td>
			<td>UCT Specialties, LLC</td>
			<td>A0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor ixon Ultra DU 897V EMCCD</td>
			<td>Andor</td>
			<td>DU-897U-CSO-#BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Solis software</td>
			<td>Andor</td>
			<td></td>
			<td>For controlling the Andor EMCCD</td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>532/640/25</td>
			<td></td>
		</tr>
		<tr>
			<td>Band-pass filter</td>
			<td>Chroma</td>
			<td>NF03-405/488/532/635E-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>Biotin-PEG-SVA</td>
			<td></td>
		</tr>
		<tr>
			<td>Coenzyme A free acid</td>
			<td>Prolume</td>
			<td>309-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Coolterm software</td>
			<td></td>
			<td></td>
			<td>For controlling the syringe pump</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>For controlling the microscope, camera, stage, and pump.</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Semrock</td>
			<td>R405/488/532/635</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA microprep 50RNX</td>
			<td>Fisher Scientific</td>
			<td>NC1139450</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Devcon</td>
			<td>14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Firefly luciferin D-Luciferin free acid</td>
			<td>Prolume</td>
			<td>306-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>BP1185500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen perioxide</td>
			<td>Sigma-Aldrich</td>
			<td>216763-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Olympus</td>
			<td>Z-81226A</td>
			<td>Low auto-fluorescence</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E1500</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGASCRIPT T7 Transcription Kit</td>
			<td>Thermo fisher</td>
			<td>AM1334</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>MMX04751</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Scientific</td>
			<td>3048</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-FLAG M2-Cy3</td>
			<td>Sigma-Aldrich</td>
			<td>A9594</td>
			<td></td>
		</tr>
		<tr>
			<td>mPEG-SVA</td>
			<td>Laysan Bio Inc</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>MS(PEG)4</td>
			<td>Thermo Scientific</td>
			<td>22341</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5M)</td>
			<td>Thermo Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>No 1.5 microscope Cover glass</td>
			<td>Fisherband</td>
			<td>12-544-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Laser, 532nm 100mM</td>
			<td>Olympus digital Laser system</td>
			<td>CMR-LAS 532nm 100mW</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus TirfCtrl software</td>
			<td>Olympus</td>
			<td></td>
			<td>For controlling the laser intensity and incident angle</td>
		</tr>
		<tr>
			<td>Optical table</td>
			<td>TMC vibration control</td>
			<td>63-563</td>
			<td>With vibration isolation</td>
		</tr>
		<tr>
			<td>Phenol chloroform isoamyl alcohol mix</td>
			<td>Sigma-Aldrich</td>
			<td>77617-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce RNA 3&#39; End Biotinylation Kit</td>
			<td>Thermo Scientific</td>
			<td>20160</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide pellets</td>
			<td>Sigma-Aldrich</td>
			<td>P1767-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior motorized XY translation stage</td>
			<td>Prior</td>
			<td>PS3J100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior PriorTest software</td>
			<td>Prior</td>
			<td></td>
			<td>For controlling the Prior motorized stage</td>
		</tr>
		<tr>
			<td>Recombinant RNasin RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top Incubator</td>
			<td>In vivo scientific (world precision Instruments)</td>
			<td>98710-1</td>
			<td>With a custom built acrylic cage</td>
		</tr>
		<tr>
			<td>Staining jar</td>
			<td>Fisher Scientific</td>
			<td>08-817</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo Scientific</td>
			<td>43-4301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>A300212</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR green II</td>
			<td>Fisher Scientific</td>
			<td>S7564</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>1725RN</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard apparatus</td>
			<td>55-3333</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (1M), pH = 7.0</td>
			<td>Thermo Scientific</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Bath</td>
			<td>Branson</td>
			<td>CPX1800H</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccinia Capping system</td>
			<td>New England Biolabs</td>
			<td>M2080S</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymo-Spin IC Columns</td>
			<td>Zymo Research</td>
			<td>C1004</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro single-molecule imaging assay for the analysis of cap-dependent translation kinetics

캡 의존성 단백질 합성은 진핵 세포에서 주요 번역 경로입니다. 다양한 생화학 적 및 유전 적 접근은 캡 의존 성 번역 및 규제의 광범위한 연구를 허용했지만,이 번역 경로의 고해상도 운동 특성화는 여전히 부족합니다. 최근에는 단일 분자 분해능으로 캡 의존형 번역 운동학을 측정하기 위한 체외 분석서를 개발했습니다. 상기 분석법은 초기 에피토프 태그폴리펩티드에 형광표지 항체 결합을 기반으로 한다. 초기 펩타이드-리보솜-mRNA 복합체를 통해 항체의 결합 및 해리를 이미징함으로써 개별 mRNA에 대한 번역 진행을 추적할 수 있다. 여기서는 mRNA 및 PEGylated 슬라이드 제제, 번역의 실시간 이미징 및 단일 분자 궤적 분석을 포함한 이 분석 방법을 확립하기 위한 프로토콜을 제시합니다. 이 분석은 개별 캡 종속 번역 이벤트를 추적할 수 있게 해주며 개시 및 신장 속도와 같은 주요 번역 역학을 해결합니다. 분석은 뚜렷한 번역 시스템에 널리 적용될 수 있으며 캡 의존성 번역 역학 및 번역 제어 메커니즘의 체외 연구에서 광범위하게 이점을 얻을 수 있습니다.

설명된 프로토콜에 따라 3'엔드 테더드 리포터 mRNA(그림1)의활성 세포 무분별 번역 동안 단일 분자 분해능을 가진 초기 N 단말 태그 폴리펩티드와개별 항체 상호작용의 이미징을 가능하게 한다. 최소 데모 실험은 3개의 합성 mRNA를 사용하여 보고됩니다: LUC (태그되지 않은 루시파라아제를 인코딩), LUCFLAG (3xFLAG 태그 루시파라아라기), 마력-LUC<...

Watch this Scientific Journal Video about An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics at JoVE.com

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

개별 번역 이벤트를 추적하면 캡 종속 번역 메커니즘에 대한 고해상도 운동 연구를 수행할 수 있습니다. 여기서 우리는 형광으로 표지된 항체와 에피토프 태그가 달린 초기 성 펩티드 사이의 화상 진찰 상호 작용에 근거를 둔 체외 단일 분자 분석체를 보여줍니다. 이 방법은 활성 체외 캡 의존 변환 동안 개시 및 펩티드 신장 운동의 단일 분자 특성화를 가능하게한다.

캡 의존형 번역 역학 분석을 위한 생체외 단일 분자 이미징 분석

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

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Research

JoVE Journal

Biochemistry

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

3.0K 조회수.  Memorial Sloan Kettering Cancer Center. 개별 번역 이벤트를 추적하면 캡 종속 번역 메커니즘에 대한 고해상도 운동 연구를 수행할 수 있습니다. 여기서 우리는 형광으로 표지된 항체와 에피토프 태그가 달린 초기 성 펩티드 사이의 화상 진찰 상호 작용에 근거를 둔 체외 단일 분자 분석체를 보여줍니다. 이 방법은 활성 체외 캡 의존 변환 동안 개시 및 펩티드 신장 운동의 단일 분자 특?...

비디오: An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

소설 형광 프로브와 플라즈마 막에서 포스파티딜 이노시톨 -4,5- 비스 포스페이트의 단일 분자 슈퍼 해상도 이미징

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

연구

생화학

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

번역 개시 포유류 mRNAs에 복잡 한 대형의 Toeprinting 분석

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

단일 분자 힘 분광학에 대 한 단백질의 화학식 동원 정지

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

단일 분자 수준에서 동질성을 라벨의 프로테옴 와이드 정량화

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

단일 분자 관찰을 위한 DNA 종이 접기 나노 구조에 다인 및 키네신 모터 앙상블 생산

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

단일 분자 물고기에 의한 전사 활성 알레일의 단일 세포 분석

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

DNA에 조립되는 EWS-FLI1 응축수의 단일 분자 이미징

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

단백질 인산화의 정량화를 위한 최적화된 단일 분자 풀다운 분석

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

완전히 재구성된 Cdc45/MCM2-7/GIN(CMG)의 모션 이미징

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

저자 스포트라이트: Investigating the motion Dynamics of the eukaryotic replisome components at the single-molecule level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

완전히 재구성된 CMG의 움직임을 이미지화하기 위한 하이브리드 앙상블 및 단일 분자 분석

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Methods Article

캡 의존형 번역 역학 분석을 위한 생체외 단일 분자 이미징 분석