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Biology

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Published: June 04, 2019
doi:
10.3791/58545
, , ,
1Biological Sciences Department Hunter College,City University of New York, 2Program in Molecular, Cellular, and Developmental Biology,Graduate Center, City University of New York

Summary

在这里,我们提出了一个协议,用于可视化,检测,分析和跟踪内源性mRNA贩运在活的果蝇黑色素细胞卵室使用分子信标,旋转盘共聚焦显微镜,和开源分析软件。

Abstract

基于荧光的成像技术,结合光显微镜的发展,彻底改变了细胞生物学家进行活细胞成像研究的方式。自从开创性研究将位点特异性mRNA定位与基因表达调控联系起来以来,检测RNA的方法已经大大扩展。动态 mRNA 过程现在可以通过检测 mRNA 的方法进行可视化,再加上足够快的显微镜设置,可以捕获分子行为的动态范围。分子信标技术是一种基于杂交的方法,能够直接检测活细胞中的内源性转录。分子信标是发夹状、内部淬火、单核苷酸鉴别核酸探针,只有在杂交到独特的靶序列时才会产生荧光。当与先进的荧光显微镜和高分辨率成像相结合时,它们使人们能够对mRNA的细胞内运动进行空间和时间跟踪。虽然这项技术是能够检测内源转录本的唯一方法,但由于在设计活细胞成像的此类探针方面有困难,细胞生物学家尚未完全接受这项技术。一种新的软件应用程序PinMol,允许增强和快速设计的探头最适合有效地杂交到活细胞内的mRNA目标区域。此外,高分辨率、实时图像采集和当前开源图像分析软件可实现精细的数据输出,从而更精细地评估 mRNA 生命周期中涉及的动态过程的复杂性。

在这里,我们提出了一个全面的协议,用于设计和交付分子信标到果蝇黑色素细胞的卵子室。通过旋转盘共聚焦显微镜对内源性母体mRNA进行直接和高度特异性的检测和可视化。成像数据使用冰面软件中的物体检测和跟踪进行处理和分析,以获取有关 mRNA 动态移动的详细信息,mRNA 被传输并本地化到卵母细胞内的专门区域。

Introduction

通过开发基于荧光的活细胞成像技术,能够用空间和时间分辨率来可视化动态事件。目前,在体内mRNA可视化是通过基于RNAaptamer-蛋白相互作用、RNAaptamer诱导的有机染料荧光和核酸探退火1、2、3.均提供高特异性、灵敏度和信背景比。然而,以RNA贴合剂为中心的方法需要大量的基因操作,其中转基因被设计为表达具有蛋白质或有机染料结合所需的人工结构图案的RNA。例如,MS2/MCP 系统需要共同表达一个转基因表达的RNA结构,该结构包含噬菌体 MS2 涂层蛋白 (MCP) 的多重串联重复,另一个转基因编码荧光蛋白融合到MCP4,5。在RNA中加入这种二次结构图案,以及大量荧光标记的蛋白质,引起了人们的担忧,即原生RNA过程可能受到影响6。一项解决了这一问题并提供额外独特优势的技术是核酸方法,分子信标(MB)。MB允许多向地源性mRNA检测,区分单核苷酸变异,以及快速动力学与目标mRNA7,8杂交。MB是寡核苷酸探针,一旦杂交到目标(图1C)9,在经历氟性形成变化之前,它们仍停留在淬火发夹褶皱中。几个组已经成功地使用MB检测非编码RNA(微RNA和lncRNA)10,11,12,13,RNA逆转录病毒14和动态DNA蛋白交互15.它们已成功应用于各种生物体和组织成像,如斑马鱼胚胎16、神经元13、肿瘤组织17、区分心肌细胞18和沙门氏菌19.

在这里,我们描述了活D.melanogaster卵室内源性mRNA的设计、交付和检测方法,以及足够快的显微镜设置,能够捕获活性分子传输的动态范围。D. melanogaster卵子室是一个理想的多细胞模型系统,用于广泛的发育研究,从早期生殖系干细胞分裂和母体基因表达到分段体计划20的生成, 21.蛋室易于隔离,大而半透明,能够承受数小时的体外分析,使其高度易于成像实验。在积极翻译之前,许多工作都集中在母体抄本到离散亚细胞区域的不对称本地化上。特别是,oskar mRNA定位及其随后在卵母细胞后极的翻译必须以严格调节的方式发生,以避免致命的双体胚胎表型22。oskar mRNA被转录在15个生殖系细胞中,称为护士细胞,并通过细胞质桥(称为环管)积极输送到卵母细胞,即成为成熟卵子并最终受精的生殖细胞(图1A)).现有大量关于蛋白质因子动态招募和交换的信息,以及其远程细胞内旅行,使oskar成为学习的首选mRNA生命周期的许多过程。MB有助于揭示mRNA定位过程的细节,并破译控制果蝇细胞生成过程中控制mRNA传输的蛋白质因子的调控和功能。特别是,通过将MB微注射到护士细胞中并进行活细胞成像实验,可以追踪内源性mRNA8,23。

此处提供的路线图提供了完整流程的步骤,从使用 MB 进行活细胞成像实验、获取成像数据,到执行数据分析以跟踪其原生细胞环境中的内源性 mRNA。可以修改和进一步优化这些步骤,以满足在他们自己的实验室环境中处理其他组织/细胞类型的研究人员的需求。

Protocol

1. 活细胞成像MB的设计 折叠目标RNA序列,使用mfold服务器(http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form)中的”RNA形式”预测mRNA目标的二次结构。 以 FASTA 格式粘贴/上传目标序列,选择 5 或 10% 次优极性(自由能量在 MFE 值的 5 或 10% 以内折叠的结构),并相应地调整计算折叠的最大数量(例如,较大的 10%次优度)。注意:在设计MB时,包括次优二级结构,可以识别目标mRNA内可能比?…

Representative Results

使用PinMol,可以为一个 mRNA 目标设计多个 MB(图 1B-C)。经过合成和纯化,使用体外分析对选定的MB进行特征描述和比较。 图1:内源性mRNA活细胞成像技术和组织描述。(A) 用于显微注射的中期果蝇?…

Discussion

果蝇卵室内源性mRNA贩运的实时可视化依赖于使用特定、高效且耐核酸酶的MB,现在可以使用PinMol软件轻松设计。MB 是特定探针,旨在检测目标 mRNA 内的独特序列(最好是无二次结构的区域),从而能够对转录记录进行高解析检测。在其他组织/细胞类型采用此技术/协议时,唯一的限制是感兴趣的样本的 MB 交付效率。虽然其他方法要求对组织进行基因操作,以表达贴合剂和RNA结合蛋白,并带有荧?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Salvatore A.E.Marras(罗格斯大学公共卫生研究所中心)对分子信标的合成、标记和纯化,并感谢丹尼尔·圣约翰斯顿(剑桥大学古登研究所)的奥斯卡-MS2/ MCP-GFP转基因苍蝇库存。这项工作得到了国家科学基金会CAREER奖1149738和专业员工大会-CUNY奖的支持。

Materials

Spectrofluorometer Fluoromax-4 Horiba-Jobin Yvon n/a Photon counting spectrofluorometer
Quartz cuvette Fireflysci (former Precision Cells Inc.) 701MFL
Dumont #5 tweezer World Precision Instruments 501985 Thin tweezers are very important to separate out the individual egg chambers
Halocarbon oil 700 Sigma-Aldrich H8898
Cover slip No.1 22 x 40mm VWR 48393-048
Dissecting microscope Leica MZ6 Leica Microsystems Inc. n/a
CO2 fruit fly anesthesia pad Genesee Scienific 59-114
Tris-HCL pH7.5 Sigma-Aldrich 1185-53-1
Magnesium chloride Sigma-Aldrich 7791-18-6
NaCl Sigma-Aldrich 7647-14-5
Spinning disc confocal microscope Leica DMI-4000B inverted microscope equipped with Yokogawa CSU 10 spinning disc Leica Microsystems Inc. n/a
Hamamatsu C9100-13 ImagEM EMCCD camera Hamamatsu n/a
PatchMan NP 2 Micromanipulator Eppendorf Inc. 920000037
FemtoJet Microinjector Eppendorf Inc. 920010504
Injection needle: Femtotips II Eppendorf Inc. 930000043
Loading tip: 20ul Microloader Eppendorf Inc. 930001007
Micro Cover glasses no. 1 or 1.5, 22x40mm VWR 48393-026; 48393-172
Dry yeast Any grocery store n/a
Computer, > 20 GB RAM Although processing can be carried out on most computers, higher capabilities will increase the speed of the processing
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Tags

MRNADrosophila MelanogasterEgg ChambersRNA LocalizationLive ImagingMolecular BeaconsMicroinjectionRNA BiologyReal-time VisualizationRNA TraffickingTherapeutic TargetsZebrafishCell CultureMicroscopyImage Analysis

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Catrina, I. E., Bayer, L. V., Omar, O. S., Bratu, D. P. Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers. J. Vis. Exp. (148), e58545, doi:10.3791/58545 (2019).
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