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.

마우스 조직을 얻기 위해 사용되는 사례가 트 렌토 대학의 동물의 보호 (OPBA) (이탈리아)의 몸에 의해 승인 된 프로토콜 없음. 04-2015, 같은 art.31 당 입법 법령 없음. 2,014분의 26. 모든 마우스는 통합적인 생물학 센터 (CIBIO), 트 렌토 대학, 이탈리아의 모델 생물의 시설 유지했다. 주의 : 샘플 중 RNA의 분해를 방지하는 RNase 오염을 최소화 DEPC 처리 된 물을 사용하는 모든 버퍼를 제조 하였다. ?...

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DNA의 구조가 전사 및 염색질의 조직에서 유전자 발현의 전사 조절에 대한 우리의 이해를 발전와 프로세스를 설명하는 데 가장 중요한 것으로 입증 된 바와 같이, 이는 진실 이해력을 향상 polysom​​es의 조직 및 구조를 분석하는 것이 필수적이다 번역 및 규제. 전술 한 프로토콜에 부드럽게 평평한 표면에 고정화 polysom​​es의 최적 밀도는 AFM 영상 적합한 얻는다. 편리하게 ?...

단백질의 합성은 1,2- 세포에서 가장 에너지 소비 프로세스이다. 그러므로, 단백질 존재비 주로 병진 아닌 전사 수준 3,4,5-에서 제어된다는 의외이다. polysom​​e 기능성 단백질 판독에 mRNA의 정보를 변환하는 기본적인 거대 분자 구성 요소입니다. Polysomes는 지금까지 여러 번역 컨트롤 6-13을 수렴 거대 분자 복합체로 인식하고 있습니다. 리보솜 구조 14- (16)에 대한 연구 수백에도 불구하고, 번역의 역학에 대한 자세한 분자 통찰력과 polysomes의 토폴로지는 제한된이자가 발생했습니다. 결과적으로, 기본 polysom​​al의 리보 핵산 단백질 복합체의 조직과 번역에 대한 잠재적 효과는 여전히 다소 애매한 문제입니다. Polysom​​es 잠재적 무엇 뉴 클레오 및 후생 유전 학적 contro 미러링, 여전히 알 수없는 주문 및 기능 조직을 숨길 수 있습니다LS는 전사 필드에 표시했다. 사실,이 흥미로운 가설의 조사 추가 연구와 새로운 기술 접근​​ 방식이 필요합니다. 이 라인에서, 구조 기술과 원자 힘 현미경 fruitfully 마찬가지로 뉴 클레오 17, 18 달성 한 것에, 유전자 발현을 제어하기위한 새로운 메커니즘을 해명하기 위해 협력 할 수 있습니다. 리보솜 14-16의 발견 이래, 그 구조는 광범위 단백질 합성의베이스 메커니즘의 분자에 대한 설명을 제공 원핵 19,20, 최근에는 인간 (22) 효모 21 특징으로하고있다. Polysomes는 초기 일반적인 2D 형상기구 (14)를 형성하는, 소포체의 막에서 인식되었다. 앞서 언급 한 바와 같이, polysom​​e 조립체 리보솜 구조로서 지속적인 관심의 대상이 아니었다. 과거 polysom​​es TR은 본질적으로 연구되어왔다ansmission 기술을 EM은 기반. 최근에, 냉동-EM 기술은 체외 번역 시스템 23-26에서 정제 polysomes의 3D 복원, 사람 세포 용 해물 (27) 또는 셀 (28)의 수있었습니다. 이러한 기술은 polysomes 23, 26 ~ 28과 밀 배아 polysomes (24)에 인접 리보솜의 접촉면의 예비 분자 설명에 리보솜 - 리보솜 조직에 대한 더 정제 된 정보를 제공했다. 따라서, 냉동-EM 단층 촬영 분자 세부 리보솜 - 리보솜 상호 작용의 공개를 할 수 있지만 광범위한 후 처리 부담하고 재건 데이터를 처리하기위한 무거운 전산 자원을 필요로하는 분석한다. 또한, 리보솜 리보솜 작용의 분자 세부 수득 리보솜의 고해상 맵이 필요하며, 기준 리보솜이 종류는 몇 종 가능하다. 중요한 크라이 EM 단층 무연 RNA를 검출 할 수 없다. Theref철광석은 새로운 기술은 완전히 변환 장치의 구성을 이해하기 위해 필요하다. 냉동-EM 옆에, AFM은 낮은 진핵 생물 29-33과 인간 (27)에 polysomes 직접 이미징을위한 유용한 도구로 사용되어왔다. EM에 비해 AFM은 샘플 고정 또는 라벨이 필요하지 않습니다. 또한, 측정 값에 가까운 생리적 조건에서 명확 리보솜 벗은 RNA 가닥 (27)을 모두 식별하는 고유의 가능성을 행할 수있다. 단일 polysom​​es의 영상은 광범위하고 무거운 후 처리에 재건은 냉동-EM 현미경에 의해 요구 분석에 비해 조금 후 처리 노력으로 나노 해상도에서 이미지의 수천을 획득, 상대적으로 빠르게 수행 할 수 있습니다. 따라서, AFM 데이터 처리 및 분석은 고가의 워크 스테이션과 높은 컴퓨팅 파워를 필요로하지 않습니다. 이와 같이,이 기술은 polysom​​al 도형에 대한 정보, 형태 학적 특성 (예 : 수집높이, 길이 및 폭), 리보솜 밀도없는 RNA의 존재 및 극저온 EM보다 높은 처리량 polysome 당 27 리보솜의 수. 이러한 방식으로, AFM은 polysomes (27)을 묘사하는 EM 기술에 대한 강력하고 상호 보완적인 접근 방식을 나타냅니다. 여기에서 우리는 정화의 AFM이 이미지에 적용하고 마우스 뇌 polysom​​es을 분석 데이터 분석에 대한 완전한 파이프 라인을 제시한다. 제안 된 프로토콜은 정화 문제와 AFM 이미징에 사용되는 운모 기판에 polysom​​es의 정확한 증착에 초점을 맞추고있다. 쉽게 AFM 커뮤니티에서 사용되는 일반적인 소프트웨어와 함께 수행 될 수있는 종래의 입자 분석 외에도 RiboPick라는 ImageJ에 플러그 (34)는, polysome 27, 35 당 리보솜 수를 계수되게된다.

<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

병진 기계, 즉, polysome 또는 polyribosome은 세포에서 가장 크고 가장 복잡한 세포질 기계 중 하나입니다. 리보솜, mRNA를 여러 단백질과 비 코딩 RNA를 형성 Polysom​​es는 번역 컨트롤이 일어날 플랫폼을 통합 나타냅니다. 리보솜 널리 연구되고있다 동안 그러나, polysom​​es의 조직은 여전히​​ 포괄적 인 이해가 결여되어있다. 따라서 많은 노력이 polysom​​e 조직과 내장 될 수있다 병진 제어의 새로운 메커니즘을 규명하기 위해 필요합니다. 원자력 현미경 (AFM)을 나노 스케일 해상도에서 3D 영상의 획득이 가능 사형 프로브 현미경의 유형이다. 전자 현미경 (EM) 방법에 비해, AFM의 주요 이점 중 하나는, 공기와 용액 중의 두 이미지 수천 취득 시료를 활성화 할 수 있다는 것이다은 염색 친 고정 할 필요없이 주변의 생리적 조건 하에서 유지 될cedures. 여기서, 마우스 뇌 운모 기판에 자신의 증착에서 polysom​​es의 정확한 정제 상세한 프로토콜 설명한다. 이 프로토콜은 AFM 및 3 차원 물체 그들의 재건과 공기와 액체의 polysom​​e 촬영을 할 수 있습니다. 극저온 전자 현미경 (EM 극저온) 상보는 제안 된 방법은 편리 체계적 polysom​​es를 분석하고 조직을 연구에 사용될 수있다.

전체 마우스 뇌의 자당 그라데이션 polysomal 프로파일 그들의 무게와 크기에 따라 거대 분자를 분리 polysom​​al 프로파일 링하여 세포 용 해물에서 polysom​​es을 정화 할 수있다. polysom​​al 프로파일로, 이러한 예에서와 같이 배양 된 세포 또는 조직에서 얻은 세포질 해물은 (자신의 침강 계수에 따라 40S와 60S 서?...

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.

원자 힘 현미경과 뇌 Polysom​​es에서 피어링

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.

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In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Vivo에서 단일 분자 초파리 연 접 모터 신경 맨끝에 추적

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

A Novel In Vitro Model of Blast Traumatic Brain Injury

모델을 새로운 시험관에 폭발 외상 성 뇌 손상의

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

원자 힘 현미경과 뇌 Polysom​​es에서 피어링

원자 힘 현미경과 뇌 Polysomes에서 피어링