この研究は、国立衛生研究所[R01GM121847]によってサポートされました。メモリアルスローンケタリングがんセンター(MSKCC)サポートグラント/コアグラント(P30 CA008748);そしてMSKCC機能ゲノミクスイニシアティブ

著者らは開示するものは何もない。

一般的なインビトロTIRF単一分子実験と比較して、ここで説明するアッセイによる単一分子イメージングは、細胞抽出物および蛍光標識抗体の高濃度の使用による追加的に複雑である。1ラウンドの表面PEGylationの一般的な手法と比較して、PEGylationの第2ラウンド(ステップ2)は、検出面15に結合する非特異的抗体を大幅に減少させる。蛍光性抗体の拡散濃度が高いと、極め...

真核生物系における翻訳は、主に7-メチルグアノシン(m7G)キャップ依存経路1を介して起こる。研究は、真核生物翻訳の開始段階がレート制限であり、規則2、3、4の共通の目標であることを示している。キャップ依存性翻訳のメカニズムは、遺伝的5、生化学的6、7、8、構造9、およびゲノム10バルクアプローチを用いて広範囲に研究されてきた。これらの方法は、キャップ依存の開始を制御する多様なメカニズムを特定しましたが、その解決は、異種および非同期開始イベントからの信号のアンサンブル平均に制限されます。さらに最近では、生体内翻訳イベントの個々の可視化は、新生ポリペプチド11、12、13、14上のエピトープに結合する蛍光抗体を測定する方法によって可視化されている。しかし、これらの新しいアプローチは、複数の蛍光抗体が新生ペプチドに結合して、単一の翻訳事象を高細胞内蛍光のバックグラウンドから解決できるようにするために、個々の開始事象を解決する能力にも限界があります。多くの生物学的相互作用において、解決された個々の運動事象は、分子レベルで同期することができない複雑な多段階および反復的な生物学的プロセスを理解するための重要な洞察を提供してきた。キャップ依存の開始と規制をより深く理解するためには、個々の翻訳イベントのダイナミクスを追跡できる新しい方法が必要です。
我々は最近、単一分子分解能15を有するキャップ依存性開始運動を測定するインビトロアッセイを開発した。この開始経路3,16に関与する既知および未知のタンパク因子の多数を考慮すると、単一分子アッセイは、細胞因子および堅牢な翻訳活性17、18、19、20、21、22、23、24、25の保存の恩恵を受けるために、既存のインビトロ無細胞翻訳系と互換性があるように開発された。さらに、無細胞翻訳システムを使用することで、単一分子観測と以前のバルク結果との比較がより互換性があります。このアプローチは、キャップ依存開始の既存の機械化フレームワークに新しい単一分子運動学的洞察を簡単に統合することを提供する。単一分子アッセイを確立するために、従来の無細胞翻訳システムは3つの方法で修飾されます:エピトープコード配列はレポーターmRNAのオープンリーディングフレーム(ORF)の最初に挿入されます。レポーターmRNAの3′末端は、単一分子検出表面へのmRNA末端テザリングを容易にするためにビオチン化される。蛍光標識抗体は翻訳抽出物に補足されます。これらの修飾は、基本的な分子生物学の技術と一般的に入手可能な試薬のみを必要とします。さらに、これらの改変および単一分子イメージング条件は、バルク無細胞翻訳反応15の翻訳動態を維持する。
このアッセイ(図1)において、5′末端キャップおよび3′末端ビオチン化レポーターmRNAは、流れチャンバ内のストレプトアビジン被覆検出面に固定化される。流れチャンバは、次いで蛍光標識抗体を補充した無細胞翻訳混合物で満たされる。エピトープ配列26,27の下流にある約30~40コドンに対するmRNA翻訳が行われた後、エピトープはリボソーム出口トンネルから出現し、蛍光標識抗体と相互作用するようになります。この相互作用は迅速であり、単一分子蛍光イメージング技術による検出により、活性無細胞翻訳中の単一分子分解能による翻訳キネティックスの追跡が可能になります。このアッセイは、キャップ依存翻訳キネティクスとその規制、特に働くバルクインビトロアッセイを有するシステムに関するインビトロ研究に大きく利益をもたらすべきである。
この単一分子アッセイを確立するための前提条件は、働くバルク無細胞翻訳アッセイであり、市販または前に述べた方法28に記載された翻訳抽出物を使用して達成することができる。真核生物翻訳抽出物は、真菌、哺乳類、および植物28を含む多様な細胞から得ることができる。イメージングのために、このアッセイは、調整可能なレーザー強度と入射角度、電動サンプルステージ、電動流体システム、およびサンプル温度制御装置を備えたTIRF顕微鏡を必要とします。このような要件は、現代 のインビトロ 単一分子TIRF実験に対して一般的であり、異なる方法で達成され得る。ここで紹介する実験では、市販の顕微鏡、ソフトウェア、および付属品から構成される客観的なタイプのTIRFシステムを使用して、すべての 材料表に記載されています。

<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

キャップ依存性タンパク質合成は、真核細胞における主要な翻訳経路である。様々な生化学的および遺伝的アプローチにより、キャップ依存翻訳とその調節に関する広範な研究が可能になっているが、この翻訳経路の高解像度運動特性は依然として欠けている。最近では、キャップ依存性翻訳キネティクスを単一分子分解能で測定する インビトロ アッセイを開発しました。このアッセイは、蛍光標識された抗体が、新生エピトープタグ付きポリペプチドに結合することに基づいている。新生ペプチド-リボソーム-mRNA複合体との抗体の結合および解離をイメージングすることにより、個々のmRNA上の翻訳進行を追跡することができます。ここでは、mRNAおよびPEGylatedスライド調製物、翻訳のリアルタイムイメージング、単一分子軌道の解析など、このアッセイを確立するためのプロトコルを提示する。このアッセイは、個々のキャップ依存翻訳イベントの追跡を可能にし、開始率や伸長率などの主要な翻訳キネティクスを解決します。このアッセイは、明確な翻訳システムに広く適用することができ、キャップ依存翻訳キネティクスおよび翻訳制御機構の インビトロ 研究に広く役立つべきである。

記載されたプロトコルに従って、3'末端テザリングレポーターmRNAの活性無細胞翻訳中に単一分子分解能を有する新生N末端タグ付きポリペプチドとの個々の抗体相互作用のイメージングを可能にする(図1)。最小の実証実験は、3つの合成mRNAを使用して報告されます: LUC (タグなしルシファーゼをコード化), LUCFLAG (3xFLAGタグ付きルシファーゼをコ?...

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

個々の翻訳イベントを追跡することで、キャップ依存翻訳メカニズムの高解像度の運動研究が可能になります。ここでは、蛍光標識抗体とエピトープタグ付き新生ペプチドとのイメージング相互作用に基づく インビトロ 単分子アッセイを示す。この方法は、 インビトロ キャップ依存的なアクティブな翻訳中の開始およびペプチド伸長性キネティクスの単一分子特性を可能にする。

キャップ依存翻訳キネティクス解析のための インビトロ 単分子イメージングアッセイ

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

Research

JoVE Journal

Biochemistry

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

3.0K ビュー.  Memorial Sloan Kettering Cancer Center. 個々の翻訳イベントを追跡することで、キャップ依存翻訳メカニズムの高解像度の運動研究が可能になります。ここでは、蛍光標識抗体とエピトープタグ付き新生ペプチドとのイメージング相互作用に基づく インビトロ 単分子アッセイを示す。この方法は、 インビトロ キャップ依存的なアクティブな翻訳中の開始およびペプチド伸長性キ?...

ビデオ: 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.

新規蛍光プローブと形質膜におけるホスファチジルイノシトール4,5-ビスリン酸の単分子超解像イメージング

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

生化学

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.

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

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.

力の単一分子分光法のための蛋白質の共有結合固定

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.

プロテオーム的分類の単一分子レベルでの均一性の定量化

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.

単一分子観測のためのDNA折り紙ナノ構造に関するダインとキネシンモーターアンサンブルの製造

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.

単一分子FISHによる転写活性アレスの単一細胞解析

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.

DNA上に集合したEWS-FLI1凝縮体の1分子イメージング

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.

タンパク質リン酸化の定量のための最適化された1分子プルダウンアッセイ

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

完全再構成されたCdc45/MCM2-7/GINS(CMG)の運動のイメージング

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.

著者スポットライト:真核生物のレプリソーム成分の運動ダイナミクスを1分子レベルで調査

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.

完全に再構成されたCMGの動きを画像化するためのハイブリッドアンサンブルおよび単一分子アッセイ

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

キャップ依存翻訳キネティクス解析のための インビトロ 単分子イメージングアッセイ

キャップ依存翻訳キネティクス解析のためのインビトロ単分子イメージングアッセイ