Name: peering at brain polysomes with atomic force microscopy
Uploaded: 2016-03-16T15:00:00
Description: Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.

Fare dokuları elde etmek için kullanılan uygulamalar Trento Üniversitesi Hayvanları Koruma (OPBA) (İtalya) için Vücut tarafından kabul edildi, protokol yok. 04-2015 olarak Madde.31 başına Kanun Hükmünde Kararname no. 26/2014. Tüm fareler Bütünleştirici Biyoloji Merkezi&#39;nin (CIBIO), Trento Üniversitesi, İtalya Model Organizma Tesisleri&#39;nde tutuldu. Dikkat: numunelerin herhangi RNA bozulmasını önlemek RNaz kontaminasyonu en aza indirmek için DEPC arıtılmış su kullanılarak tüm tamponları hazırlama...

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DNA&#39;nın yapısı transkripsiyon ve kromatin organizasyonu gen ifadesinin transkripsiyonel kontrolü anlayışımızı ilerledikçe süreci tanımlamak için büyük önem olduğu kanıtlanmış gibi, doğru anlama geliştirmek için polizom organizasyonunu ve yapısını analiz etmek esastır çeviri ve düzenleme. tarif edilen protokol ile hafifçe düz bir yüzey üzerinde immobilize polizom optimal yoğunluğu, AFM görüntüleme için uygun elde edilir. uygun hazırlanan numuneler i...

Protein sentezi hücrelerinde 1,2 en çok enerji tüketen bir süreçtir. Bu nedenle, protein bolluğu ağırlıklı öteleme ziyade transkripsiyonel seviyede 3,4,5 kontrol edilir şaşırtıcı değildir. Polizom fonksiyonel proteinli okumalarla içine mRNA bilgiyi dönüştüren temel makromoleküler bileşenidir. Polisomlar şimdiye kadar birkaç öteleme kontroller 6-13 yakınsama makromoleküler kompleksleri olarak kabul edilmektedir. Ribozom yapısı 14- 16 çalışmaların yüzlerce rağmen, çeviri dinamikleri hakkında detaylı moleküler anlayışlar ve polizom topoloji sınırlı bir ilgi ile karşılaştı. Bunun bir sonucu olarak, yerli polysomal ribonükleoprotein kompleksinin organizasyonu ve tercüme üzerindeki potansiyel etkisi hala oldukça belirsiz sorunlardır. Polisomlar potansiyel neler nükleozom ve epigenetik contro yansıtma, henüz bilinmeyen sipariş ve işlevsel organizasyonları gizleyebilirls transkripsiyon alanı için temsil etmişlerdir. Nitekim, bu ilginç hipotezin araştırılması ek çalışmalar ve yeni teknik yaklaşımlar gerektirir. Bu doğrultuda, yapısal teknikler ve atomik kuvvet mikroskopisi fruitfully benzer nükleozom 17,18 için elde ne kadar, gen ifadesini kontrol etmek için yeni mekanizmalar çözülmeye işbirliği olabilir. Ribozom 14-16 keşfinden beri yapısı yoğun protein sentezi tabanında mekanizmaların moleküler bir açıklama temin prokaryotlarda 19,20, daha yakın zamanda, insan 22 maya 21 ve karakterize edilmiştir. Polisomlar başlangıçta tipik 2B geometrik örgütleri 14 oluşturan, endoplazmik retikulum zarı üzerinde kabul edildi. Daha önce belirtildiği gibi, Polizom montaj ribozom yapısı olarak sürekli ilgi nesnesi olmamıştır. Geçmişte, Polisomlar tr esasen çalışılmıştıransmission teknikleri EM-tabanlı. Ancak son zamanlarda, kriyo-EM teknikleri in vitro çeviri sistemleri 23-26 saflaştırılmış polizom 3D rekonstrüksiyonu, insan hücre lizatları 27 veya hücre 28 sağladı. Bu teknikler polizom 23, 26-28 ve buğday tohumu polizom 24 bitişik ribozom temas yüzeylerinin bir ön moleküler tanımındaki ribozom-ribozom organizasyon hakkında daha rafine bilgi sundu. Böylece, cryo-EM tomografi moleküler detaylı ribozom-ribozom etkileşimlerin açıklanmasını sağlar, ancak kapsamlı post-processing ile yatar ve rekonstrüksiyon verileri işlemek için ağır hesaplama kaynakları gerektiren analiz eder. Ayrıca, ribozom ribozom etkileşimlerinin moleküler detay elde etmek üzere, ribozom yüksek ölçüde çözülmesi harita gereklidir, ve referans ribozom bu tür bir kaç tür için kullanılabilir. Önemlisi, cryo-EM tomografi serbest RNA tespit etmek mümkün değildir. therefCevher, yeni teknikler tamamen çeviri makine organizasyonunu anlamak için gereklidir. Cryo-EM yanında, AFM de düşük ökaryotlar 29-33 ve insanlarda 27 polizom doğrudan görüntüleme için yararlı bir araç olarak kullanılmıştır. EM ile karşılaştırıldığında, AFM hiçbir örnek tespit veya etiketleme gerektirir. Buna ek olarak, ölçümler, yaklaşık olarak fizyolojik koşullar altında ve açıkça ribozomlar çıplak RNA şeritleri 27, her iki belirleme benzersiz olasılığı ile gerçekleştirilebilir. Tek polizom görüntüleme geniş ve ağır post-processing ve yeniden cryo-EM mikroskobu ile gerekli analizleri karşılaştırıldığında küçük post-processing çabayla nano çözünürlükte görüntülerin binlerce elde edilmesi, nispeten hızlı bir şekilde yapılabilir. Sonuç olarak, AFM veri işleme ve analiz pahalı iş istasyonları ve yüksek işlem gücü ihtiyacı yoktur. Bu nedenle, bu teknik polysomal şekilleri hakkında bilgi, morfolojik özellikleri (örneğin toplaryükseklik, uzunluk ve genişlik), ribozom yoğunlukları, serbest RNA&#39;nın varlığını ve kriyo-EM daha yüksek bir verimle polisom 27 ortalama ribozom sayısı. Bu şekilde, AFM Polisomlar 27 canlandıracak EM tekniklerine güçlü ve tamamlayıcı bir yaklaşımı temsil etmektedir. Burada arınma gelen AFM görüntüye uygulanan ve fare beyin Polisomlar analiz veri analizi için tam bir boru hattı mevcut. önerilen protokol arıtma konularında ve AFM görüntüleme için kullanılan mika yüzeylerde polizom doğru birikimi üzerinde duruluyor. Kolayca AFM topluluğu tarafından kullanılan ortak yazılım ile gerçekleştirilebilir geleneksel parçacık analizine ek olarak, RiboPick adlı bir ImageJ 34 eklentisi, polisom 27 35 ortalama ribozom sayısını saymak için sunulmuştur.

<table>
	<tbody>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DNAseI</td>
			<td>Thermo Scientific</td>
			<td>89836</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>RiboLock RNAse Inhibitor</td>
			<td>Life technologies</td>
			<td>EO-0381</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma</td>
			<td>40718</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma</td>
			<td>T8532</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>Prepararation of lysate</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S5016</td>
			<td>Sucrose gradient preparation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima LE-80K</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor</td>
			<td>Beckman Coulter</td>
			<td>SW 41 Ti</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Polyallomer tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>Sucrose gradient centrifugation</td>
		</tr>
		<tr>
			<td>Density Gradient Fractionation System&#160;</td>
			<td>Teledyne Isco</td>
			<td>67-9000-176</td>
			<td>Sucrose gradient fractionation</td>
		</tr>
		<tr>
			<td>AFM</td>
			<td>Asylum Research</td>
			<td>Cypher</td>
			<td>Polysome visualization</td>
		</tr>
	</tbody>
</table>

peering at brain polysomes with atomic force microscopy

Translasyonel makine, yani, polisom ya polyribosome hücrelerde büyük ve en karmaşık sitoplazmik makine biridir. Ribozomlar, mRNA, çeşitli proteinlerin ve kodlayıcı olmayan RNA&#39;lar tarafından oluşturulan Polisomlar, translasyonel kontrol gerçekleşecek platformlar entegre temsil eder. Ribozom yaygın çalışılmıştır Ancak, polizom organizasyonu hala kapsamlı bir anlayış yoktur. Bu nedenle çok çaba polisom organizasyon ve gömülebilir translasyonel kontrol herhangi bir yeni etki mekanizmasını açıklamak için gereklidir. Atomik kuvvet mikroskobu (AFM) nano çözünürlükte 3D görüntülerin elde edilmesine izin verir tarama prob mikroskobu türüdür. Elektron mikroskopisi (EM) teknikleri ile karşılaştırıldığında, AFM ana avantajlarından biri, hava ve çözelti, iki görüntü binlerce elde örnek sağlayan olmasıdır boyama ve pro sabitlenmesi için herhangi bir gerek olmaksızın, yaklaşık olarak fizyolojik koşullar altında muhafaza edilmesiprosedürlerinin. Burada, fare beyin ve mika alt tabakalar üzerine birikmesinden polizom doğru saflaştırılması için detaylı bir protokol açıklanmaktadır. Bu protokol, AFM ve üç boyutlu nesne olarak yeniden sahip hava ve sıvı içinde polisom görüntüleme sağlar. cryo-elektron mikroskopisi (cryo-EM) Tamamlayıcı, önerilen yöntemin uygun sistematik Polisomlar analiz ve organizasyon eğitimi için kullanılabilir.

Bütün fare beyninin sukroz gradyanlı polysomal profil Kendi ağırlığı ve büyüklüğü uygun bir şekilde makromolekülleri ayıran polysomal profil ile bir selüler lizat polizom arıtmak mümkündür. polysomal profil ile, bu örnekteki gibi kültürlenen hücreler ya da dokulardan elde edilen sitoplazmik lizatları (çökelme katsayılarına göre 40S ve 60S alt birimleri, 80S ribozom ve polizom ser...

Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

Atomik Kuvvet Mikroskobu ile Beyin Polisomlar de Peering

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

The overall goal of this protocol is to provide a detailed pipeline for the accurate purification and deposition of brain polyribosomes on mica and to obtain thousands of images at the nanoscale resolution without the need for heavy post-processing or 3D reconstruction analyses. This method can help answer key questions in the fields of translation and translational control, such as understanding the impact of ribosome organization within polysomes during the rewiring of gene expression. The main advantages of this technique are that no sample fixation or labeling are required, and measurements can be carried out in near physiological conditions to easily identify the ribosomes and naked RNA stands.Although this method can provide insight into the organization of mammalian polyribosome, it can also be applied to other microorganisms, such as yeast, bacteria, and insect, as well as status involving translational controls of gene expression. Visual demonstration of this method is critical, as handling polysomal sample for absorption of mica and the washing steps are quite tricky and require handling skills and experience. Demonstrating the procedure will be Paola Bernabo, and Lorenzo Lunelli.To begin, collect and freeze brain tissue as described in the accompanying text protocol. Then, pull the frozen tissue from the freezer and bring it to the bench. Place the frozen brain tissue and liquid nitrogen into a pre-chilled mortar and pulverize it using a pestle until it forms a powder.Transfer about 25 milligrams of the brain powder to a cold microcentrifuge tube and immediately add 0.8 milliliters of lysis buffer. Pipette the mixture up and down 25 times quickly to disrupt the cells. Next, centrifuge the tube at 12, 000 x g for one minute at four degrees Celsius to pellet the cellular debris.Transfer the supernatant to a new microcentrifuge tube and keep the tube on ice for 15 minutes. Then, centrifuge the tube for five minutes to pellet nuclei and mitochondria. Next, carefully remove one milliliter from the top of a previously prepared sucrose gradient.Overlay the sucrose drop by drop with the supernatant from the cytosolic lysate. WIth the sample now loaded on top of the gradient, carefully lower the tube and a counterbalance tube into the buckets of a swinging bucket rotor. Centrifuge the gradient for 100 minutes.After ultracentrifugation, leave the tubes in their buckets for 20 minutes at four degrees Celsius, to let the gradient stabilize. Then, carefully remove the sample tube and mount it on the collector device of a density gradient fractionation system. Collect one milliliter fractions while monitoring the absorbance of nucleic acids at 260 nanometers with a UV visible light detector and put them on ice.Next, prepare 30-40 microliter aliquots of the fractions of interest, and keep them on ice. If the samples will not be used right away, store them at 80 degrees Celsius and take steps to limit the number of freeze-thaw cycles. To prepare samples for AFM imaging, first cover a washed mica sheet with 200 microliters of one millimolar nickel ii sulfate and incubate the sheet for three minutes at room temperature.Then remove the solution, dry the surface with compressed air, and place the petri dish on ice. Next, gently add the entire aliquot from the fraction of interest drop by drop on the mica. Using a 100 or 200 microliter tip, spread the sample over the entire surface of the mica, and incubate the sample on ice for three minutes.Then, cover the mica sheet drop by drop with 200 microliters of cold buffer AFM and incubate the sample on ice for one hour. Maintaining the sample at 40 degrees and using cold buffer that were prepared in RNase-free water are important to preserve polyribosome organization. Following incubation, carefully remove the buffer and wash the mica three to four times with 200 microliters of cold buffer AFM to remove any excess sucrose.Then, wash the mica three times with cold washing solution and drain the excess water from the mica using paper. The complete removal of sucrose from the absorbant samples is crucial now that you have severed both ribosome and naked RNA in the polysome. Leave the sample to dry under the chemical hood with the top of the petri dish partially opened.After two hours, close the petri dish. The sample can now be stored at room temperature. Attach the prepared sample to the sample holder of the AFM using double sided tape.Then, insert the sample holder in the AFM stage in accordance with the manufacturer's instructions. Following calibration, approach the sample until the cantilever tip engages the surface. Select a scan area of two by two microns, a resolution of at least 512 x 512 pixels, choose live background subtraction mode, and select a Z-scale of 20-25 nanometers.Then, begin image acquisition. Inspect the image, looking for the presence of round objects characterized by a height between 10 and 15 nanometers when acquiring images in the air. Next, adjust the set point and feedback parameters until sharp objects are visualized.The background should appear relatively flat in good samples with some two to four nanometer high objects. Once the image looks good, acquire several two by two micron scans at different sample areas. After the deposition of sucrose aliquots on mica, AFM provides accurate size descriptions of single polysomes that appear as clusters of tightly packed ribosomes.Taking a closer look at one of the ribosomal peaks using cross-section analysis reveals the height of ribosomal peaks are around 14 nanometers. This matches what was previously observed for human ribosomes and polysomes after air-drying. In the image on the right, a macro was used to identify ribosome location and mark each ribosome with a red circle.Using this information, the frequency distribution of the number of ribosomes per polysome can be analyzed. This experimental distribution was fitted with a Gaussian curve that was centered at 5.8 ribosomes per polysome, with a standard deviation of 1.3. Once mastered, this technique from brain pulverization to polyribosome images acquisition can be done in less than eight hours if it is performed properly.While attempting this procedure, it is important to remember to keep the sample on ice and use cold buffers for the sample deposition on the mica. Following this procedure, and using the ImageJ plugin of a developer to make available in your paper, you can precisely count the number of ribosome per polysome. After each development, this technique will pave the way to understand the kinetics of polysome formation and identifying the changes in polysome organization and their different cellular or tissue conditions.After watching this video you should have a good understanding of how to obtain thousands of polysome images for systematically analyzing and studying their organization.

Research

JoVE Journal

Neuroscience

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Nörobilim

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Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

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