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&#039; 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 ...

an-vitro-single-molecule-imaging-assay-for-analysis-cap-dependent

Universidad de Cartagena UDEC

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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

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

정량적 면역 형광 측정 글로벌 현지화 번역

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

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생화학

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9 정제 초공정 키네신-3 제품군 모터의 단일 분자 분석

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

액틴과 미세소관 커플링 다이내믹스 를 총 내부 반사 형광(TIRF) 현미경으로 시각화

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s&#8722;1 and 0.35 s&#8722;1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers

The protocol presents two methods to determine the kinetics of the fluorogenic RNA aptamers Spinach2 and Broccoli. The first method describes how to measure fluorogenic aptamer kinetics in vitro with a plate reader, while the second method details the measurement of fluorogenic aptamer kinetics in cells by flow cytometry.

플루오로겐 RNA 압타머에 대한 시험관 내 및 세포 턴온 역학 결정

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

시험관 내 전사 된 RNA-기반 루시퍼 라 제 리포터 검정은 폭스 바이러스 감염 세포에서 번역 조절을 연구 하기 위해

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

면역학 및 감염병학

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

유전자 규정의 단일 분자 이미징<em&gt; 생체에</em&gt; 절단하여 Cotranslational 인증 (CoTrAC)를 사용

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

생물학

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Micromanipulation 기법 전체적 역동성의 분석 및 Cytoskeletal 레 귤 레이 터의

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

시투 혼성화에서 2색 단일 분자 형광을 사용하여 에샤리치아 대장균의 공간과 시간에서 mRNA 운동학을 프로빙

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

세포 역학을 정량화하는 다목적 기술인 단세포 어레이(LISCA)의 라이브 셀 이미징

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...