Bu çalışma Ulusal Sağlık Enstitüleri [R01GM121847] tarafından desteklendi; Memorial Sloan Kettering Kanser Merkezi (MSKCC) Destek Hibesi/Çekirdek Hibesi (P30 CA008748); ve MSKCC Fonksiyonel Genomik İnisiyatifi.

Yazarların açıklayacak bir şeyi yok.

Tipik in vitro TIRF tek moleküllü deneylere kıyasla, burada açıklanan test ile tek moleküllü görüntüleme, hücre ekstresi ve floresan etiketli antikorun yüksek konsantrasyonu nedeniyle ek olarak karmaşıktır. Bir tur yüzey PEGylation'ın daha yaygın uygulaması ile karşılaştırıldığında, ikinci bir PEGylation turu (adım 2), algılama yüzeyine spesifik olmayan antikor bağlanmasını büyük ölçüde azaltır15. Difüzör floresan antikorların yüksek konsantrasyo...

Ökaryotik sistemlerde çeviri ağırlıklı olarak 7-metilguanosin (m7G) kap bağımlı yollar1yoluyla gerçekleşir. Çalışmalar, ökaryotik çevirinin başlangıç adımının hız sınırlayıcı ve yönetmelik2, 3,4için ortak bir hedef olduğunu göstermektedir. Kapak bağımlı çeviri mekanizmaları genetik5, biyokimyasal6, 7 ,8,yapısal9ve genomik10 toplu yaklaşımlar kullanılarak kapsamlı bir şekilde incelenmiştir. Bu yöntemler kap bağımlı başlatmayı düzenleyen çeşitli mekanizmalar tanımlamış olsa da, çözünürlükleri onları heterojen ve zaman uyumsuz başlatma olaylarından gelen sinyallerin ortalamasıyla sınırlar. Daha yakın zamanda, bireysel in vivo çeviri olayları, nascent polipeptidleri 11 , 12 , 13,14üzerindeki epitoplara floresan antikor bağlanmasını ölçen yöntemlerle görselleştirilmiştir. Bununla birlikte, bu yeni yaklaşımların bireysel başlatma olaylarını çözme yetenekleri de sınırlıdır, çünkü çoklu floresan antikorların, tek çeviri olaylarının yüksek hücre içi floresan arka plandan çözülmesine izin vermek için bir nascent peptid bağlaması gerekir. Birçok biyolojik etkileşimde, çözülen bireysel kinetik olaylar, moleküler düzeyde senkronize olması mümkün olmayan karmaşık çok noktalı ve tekrarlayan biyolojik süreçleri anlamak için kritik içgörüler sağlamıştır. Kap'a bağımlı başlatma ve düzenlemenin daha iyi anlaşılması için bireysel çeviri olaylarının dinamiklerini izleyebilecek yeni yöntemlere ihtiyaç vardır.
Yakın zamanda tek moleküllü çözünürlük15ile kap bağımlı başlatma kinetiğini ölçen bir in vitro test geliştirdik. Bu başlangıç yolu3,16'da yer alan çok sayıda bilinen ve bilinmeyen protein faktörü göz önünealındığında,tek moleküllü test, hücresel faktörlerin korunmasından ve sağlam çeviri aktivitesinden yararlanmak için mevcut in vitro hücresiz çeviri sistemleriyle uyumlu olacak şekilde geliştirilmiştir17,18,19,20,21,22,23,24,25. Ayrıca, hücresiz çeviri sistemlerinin kullanımı, tek moleküllü gözlemler ve önceki toplu sonuçlar arasında daha uyumlu karşılaştırmalar sağlar. Bu yaklaşım, yeni tek moleküllü kinetik içgörülerin, kap bağımlı başlatmanın mevcut mekanistik çerçevesine doğrudan entegrasyonunu sağlar. Tek moleküllü tahlil oluşturmak için, geleneksel hücresiz çeviri sistemi üç şekilde değiştirilir: bir muhabir mRNA'nın açık okuma çerçevesinin (ORF) başına bir epitop kodlama dizisi eklenir; muhabir mRNA'nın 3′ ucu, mRNA'nın tek moleküllü algılama yüzeyine uç bağlamasını kolaylaştırmak için biyotinillenmiştir; ve floresan etiketli antikorlar çeviri özüne desteklenir. Bu değişiklikler sadece temel moleküler biyoloji tekniklerini ve yaygın olarak bulunan reaktifleri gerektirir. Ayrıca, bu değişiklikler ve tek moleküllü görüntüleme koşulları, toplu hücre içermeyen çeviri reaksiyonlarının çeviri kinetiğini korur15.
Bu tahlilde (Şekil 1), 5′-end kapaklı ve 3′-uç biyotiniled muhabir mRNA bir akış odasında streptavidin kaplı bir algılama yüzeyine hareketsiz hale getirilir. Akış odası daha sonra floresan etiketli antikorlarla desteklenmiş hücresiz bir çeviri karışımı ile doldurulur. MRNA çevirisi epitop dizisi 26 , 27'nin aşağı akışında yaklaşık30-40kodon meydana geldiktensonra,epitop ribozom çıkış tünelinden çıkar ve floresan etiketli antikorla etkileşime girmek için erişilebilir hale gelir. Bu etkileşim hızlıdır ve tek moleküllü floresan görüntüleme teknikleri ile algılanmasını, aktif hücresiz çeviri sırasında tek moleküllü çözünürlükle çeviri kinetiğinin izlenmesini sağlar. Bu tahlil, özellikle çalışan toplu in vitro teste sahip sistemler için, kap bağımlı çeviri kinetiğinin in vitro çalışmalarına ve düzenlenmesine geniş ölçüde fayda sağlamalıdır.
Bu tek moleküllü tahlilin kurulmasının bir ön koşulu, ticari olarak mevcut olan veya daha önce açıklanan yöntemleri izleyerek hazırlanan çeviri özü kullanılarak elde edilebilen çalışan bir toplu hücre içermeyen çeviri tahlilidir28. Ökaryotik çeviri özü mantar, memeli ve bitki28dahil olmak üzere çeşitli hücrelerden elde edilebilir. Görüntüleme için, bu test ayarlanabilir lazer yoğunluğu ve olay açısı ile donatılmış bir TIRF mikroskobu, motorlu bir numune aşaması, motorlu akışkanlar sistemi ve numune sıcaklık kontrol cihazı gerektirir. Bu gereksinimler modern in vitro tek moleküllü TIRF deneyleri için geneldir ve farklı şekilde elde edilebilir. Burada sunulan deney, malzeme tablosundalistelenen ticari olarak kullanılabilen mikroskop, yazılım ve aksesuarlardan oluşan objektif tip bir TIRF sistemi kullanır.

<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

Kap bağımlı protein sentezi ökaryotik hücrelerde baskın çeviri yoludur. Çeşitli biyokimyasal ve genetik yaklaşımlar, kap bağımlı çeviri ve düzenlemesinin kapsamlı çalışmalarına izin vermiş olsa da, bu çeviri yolunun yüksek çözünürlüklü kinetik karakterizasyonu hala eksiktir. Son zamanlarda, kap bağımlı çeviri kinetiğini tek molekül çözünürlüğü ile ölçmek için bir in vitro test geliştirdik. Test, floresan etiketli antikorun nascent epitop etiketli polipeptide bağlanmasına dayanmaktadır. Antikorların nascent peptid–ribozom-mRNA komplekslerine bağlanması ve ayrışması görüntülenerek, bireysel mRNA'lardaki çeviri ilerlemesi izlenebilir. Burada, mRNA ve PEGylated slayt preparatları, çevirinin gerçek zamanlı görüntülenmesi ve tek molekül yörüngelerinin analizi de dahil olmak üzere bu tahlilin oluşturulması için bir protokol sunuyoruz. Bu tahlil, kapak bağımlı çeviri olaylarının izlenmesini sağlar ve başlatma ve uzama oranları gibi anahtar çeviri kinetiğini çözümler. Test, farklı çeviri sistemlerine yaygın olarak uygulanabilir ve kap bağımlı çeviri kinetiği ve çeviri kontrol mekanizmalarının in vitro çalışmalarına geniş ölçüde fayda sağlamalıdır.

Açıklanan protokolün ardından, 3' son bağlı muhabir mRNA'nın aktif hücresiz çevirisi sırasında tek molekül çözünürlüğü ile nascent N-terminal etiketli polipeptitlerle bireysel antikor etkileşimlerinin görüntülenmesini sağlar (Şekil 1). Üç sentetik mRNA'nın kullanımıyla minimum bir gösteri deneyi rapor edilir: LUC (etiketlenmemiş lucifeaz kodlama), LUCFLAG (3xFLAG etiketli lucifease kodlaması) ve hp-LUC<s...

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

Bireysel çeviri olaylarını izlemek, kap bağımlı çeviri mekanizmalarının yüksek çözünürlüklü kinetik çalışmalarına izin verir. Burada floresan etiketli antikorlar ve epitop etiketli nascent peptitler arasındaki görüntüleme etkileşimlerine dayanan bir in vitro tek moleküllü test gösteriyoruz. Bu yöntem, aktif in vitro kap bağımlı çeviri sırasında başlatma ve peptit uzama kinetiğinin tek moleküllü karakterizasyonunu sağlar.

Kapak Bağımlı Çeviri Kinetiğinin Analizi için Bir In Vitro Tek Moleküllü Görüntüleme Tahlili

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

3.0K Görüntüleme.  Memorial Sloan Kettering Cancer Center. Bireysel çeviri olaylarını izlemek, kap bağımlı çeviri mekanizmalarının yüksek çözünürlüklü kinetik çalışmalarına izin verir. Burada floresan etiketli antikorlar ve epitop etiketli nascent peptitler arasındaki görüntüleme etkileşimlerine dayanan bir in vitro tek moleküllü test gösteriyoruz. Bu yöntem, aktif in vitro kap bağımlı çeviri sırasında başlatma ve peptit uzama kinetiğinin tek moleküllü karakterizasyonunu sa...

Video: 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.

Nicel ayirt ölçmek küresel çeviri lokalize

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

Biyokimya

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 Saflaştırılmış Süperprosesif Kinesin-3 Ailesi Motorlarının Tek Moleküllü Analizi

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.

Aktin ve Mikrotübül Eşleşme Dinamiğinin In Vitro Total İç Yansıma Floresan (TIRF) Mikroskobu ile Görselleştirilmesi

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.

Florojenik RNA Aptamers için In Vitro ve Hücresel Turn-on Kinetiğinin Belirlenmesi

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.

In vitro transkripsiyonu RNA tabanlı luciferase Reporter assay Poxvirus-virüslü hücrelerde çeviri Yönetmeliği çalışma

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 ...

İmmünoloji ve Enfeksiyon

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.

Gen Yönetmelik Tek molekül Görüntüleme<em&gt; In vivo</em&gt; Dilinim tarafından Cotranslational Aktivasyon (CoTrAC) kullanma

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

Biyoloji

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.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

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.

Morfogenetik dinamiklerini analiz ve hücre iskeleti düzenleyiciler ciro sağlayan Embriyolardan teknikleri

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.

Situ Hibridizasyonda İki Renkli Tek Moleküllü Floresan Kullanarak Escherichia coli'de Uzay ve Zamanda MRNA KinetikLerini Sondalama

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.

Tek Hücreli Dizilerin Canlı Hücre Görüntülemesi (LISCA) - Hücresel Kinetiği Ölçmek için Çok Yönlü Bir Teknik

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