成像核内作用棒在活热应力 果蝇 胚胎
1Integrative Molecular and Biomedical Sciences,Baylor College of Medicine, 2Department of Cell and Molecular Biology, School of Molecular and Cellular Biology,University of Illinois at Urbana-Champaign, 3Verna and Marrs McLean Department of Biochemistry and Molecular Biology,Baylor College of Medicine
Summary
该协议的目标是将罗达明结合球蛋白注射到 果蝇 胚胎中，并在热应力后将图像核作用杆组装。
Abstract
该协议的目的是可视化在热应激后在活 的果蝇黑色素酯 胚胎中组装的核内作用棒。Actin 棒是伴随人类病理（包括神经退行性疾病）的保存、诱导的阿克廷应激反应 （ASR） 的标志。以前，我们表明ASR会导致形态发生失败，降低胚胎的存活率。该协议允许在高度适应成像、遗传学和生物化学的模型系统中继续研究作用杆组装和ASR的基础机制。胚胎被收集并安装在盖片上，为注射做准备。罗达明结合球蛋白（G-actin红）被稀释并加载到微需要中。每个胚胎的中心只注射一次。注射后，胚胎在高温下孵育，然后通过分泌显微镜对核细胞内作用杆进行可视化。光漂白（FRAP）实验后的荧光恢复可以在作用杆上进行：细胞质中其他富含作用的结构也可以成像。我们发现，G-actin红 聚合物像内源性G-actin，本身并不干扰正常的胚胎发育。此协议的一个限制是，在注射过程中必须小心谨慎，以避免对胚胎造成严重伤害。然而，在实践中，将G-actinRed 注射到 果蝇 胚胎中是一种快速可靠的可视化作用杆的方法，可以很容易地用于任何基因型的苍蝇或引入其他细胞应力，包括缺氧和氧化应激。
Introduction
该协议描述了如何注射G-actinRed，以可视化在热应力胚胎中核作用杆的组装，这些胚胎正在经历不可诱导的Actin应激反应（ASR）1。我们开发了这个协议，以帮助研究ASR，在胚胎导致破坏形态生成和降低生存能力，并在成人细胞类型与病理学，包括肾衰竭2，肌肉肌病3，阿尔茨海默氏症和亨廷顿舞蹈症4，5，6，7，8。这种ASR是由许多细胞应力引起的，包括热休克9，10，11，氧化应激4，6，减少ATP合成12，和异常亨廷丁或β淀粉样寡头化4，5，6，7，9，13，14，15，16。ASR的一个标志是在受影响细胞的细胞质或细胞核中组装异常作用素棒，这是由压力引起的活性相互作用蛋白，Cofilin1，5，6，10的过度激活驱动的。不幸的是，在ASR方面仍然存在关键的知识差距。例如，行为棒的功能尚不清楚。我们不明白为什么棒在某些细胞类型的细胞质中形成，但其他细胞核。也不清楚ASR是保护性的还是对承受压力的细胞或胚胎的不适应。最后，我们仍然不知道科菲林多活化或作用杆组装背后的详细机制。因此，该方案提供了一个快速和多才多艺的检测，通过可视化活果蝇胚胎高度可处理的实验系统中的活杆形成和动力学来探测ASR。
将G-actinRed微导入活体果蝇胚胎的方案最初是为了研究组织构建事件期间正常细胞质作用素结构17的动态而开发的。在这些研究中，我们发现G-actin红注射不会对胚胎的早期发育过程产生负面影响，包括细胞因子或胃痛17，18。然后，我们修改了协议，调整了胚胎处理和G-actin红注射，以允许在进行ASR1的热应激胚胎中成像作用素棒。除G-actin红注射外，其他方法可用于在胚胎中可视化作用素。这些方法依赖于表达荧光蛋白（FPs）标记为作用素或作用素结合蛋白的领域，如营养素-mCherry、生命-生命-GFP和莫辛-GFP（在19年审查）。然而，使用这些FP探头需要谨慎，因为它们可以稳定或破坏一些行为结构，不等于标记所有行为结构20，在actin-GFP的情况下，是高度过度表达-有问题的杆组装分析，这不仅是压力依赖，而且行动浓度依赖1。因此，G-actinRed是苍蝇胚胎中棒研究的首选探针，胚胎的大尺寸允许其轻松注射。
该协议的工作流程类似于其他成熟的微注射技术，这些技术已被用于将蛋白质、核酸、药物和荧光指标注射到果蝇胚胎21、22、23、24、25、26、27。然而，在G-actinRed的微注射之后，胚胎暴露在轻度热应激下，诱导ASR和核内作用杆组装。对于能够接触苍蝇和注射钻机的实验室，这种方法应易于实施，并适应与ASR有关的具体研究路线，包括它通过不同的应力或在不同遗传背景的调制而诱导。
Protocol
1. 准备胚胎收集杯和苹果汁琼脂盘 在注射实验前五天，建造28 个或采购至少两个小胚胎收集杯。制作新鲜的60毫米苹果汁琼脂板，用于小收集杯28。在4°C时用湿纸巾覆盖的塑料盒中的存储板。注：小胚胎收集杯，填充了第1.3步描述的苍蝇数量，将提供足够的胚胎数量每个实验，同时也确保胚胎处理和注射可以在足够短的时间内完成，使早期发育阶段?…
Representative Results
图1中描绘了胚胎处理的示意图工作流程，并在表1中介绍了典型实验的时间表。一个好的实验结果的估计是，每注射10个胚胎，至少一半的被观察的胚胎将处于正确的发育阶段，没有损坏，并表现出强大的ASR与热应力在32°C。 图2A（右面板）中胚胎的代表性表面视图图像中所示，将通过核内作用素棒的组装来证明此 ASR。Actin 棒将出现在原子?…
Discussion
这种方法的意义在于，它利用了在果蝇胚胎21、22、23、24、25、26、27中建立的微射方案，使有关ASR和伴随作用杆组装的新研究得以进行。将G-actinRed注射到活胚胎中的一个主要优点是，ASR可以在各种背景下进?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
作者感谢郑柳柳和薛增辉（在索卡克实验室帮助开创这项技术）以及帮助分析的哈桑·西德（Hasan Seede）的工作。这项研究的工作由国家卫生研究院（R01 GM115111）的赠款资助。
Materials
| Adenosine triphosphate (ATP) | Millipore-Sigma | A23835G | Component of G buffer |
| Apple juice, Mott's, 64 fl oz | Mott's | 014800000344 | Component of apple juice plates |
| Bacto Agar | BD | 214010 | Component of apple juice plates |
| Bleach, PureBright Germicidal, 6.0% sodium hypochlorite | KIK International | 059647210020 | For dechorionating embryos |
| Calcium chloride | Millipore-Sigma | C1016500G | Component of G buffer |
| Cell strainer, 70 μm | Falcon | 352350 | For collecting dechorionated embryos |
| Confocal microscope, LSM 880 34-channel with Airyscan | Zeiss | 0000001994956 | For imaging intranuclear actin rods |
| Desiccant | Drierite | 24001 | For desiccating embryos |
| Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom | Zeiss | 4350649000000 | For arranging embryos on agar wedge |
| Dissecting needle, 5 in | Fisher Scientific | 08965A | For arranging embryos on agar wedge |
| Dithiothreitol (DTT) | Fisher Scientific | BP1725 | Component of G buffer |
| Double-sided Tape, Scotch Permanent, 0.5 in x 250 in | 3M | 021200010323 | For making embryo glue |
| Embryo collection cage | Genessee Scientific | 59100 | For housing adult flies and collecting embryos |
| Fine tip tweezers, Dumont Tweezer, Style 5 | Electron Microscopy Sciences | 72701D | For arranging embryos on agar wedge |
| Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm | World Precision Instruments, Inc. | TW1004 | For microneedles |
| Halocarbon oil 27 | Millipore-Sigma | H8773100ML | For hydration of embryos |
| Heated stage incubator | Zeiss | 4118579020000, 4118609020000, 4118609010000 | For confocal imaging |
| Lab Tissue Wipers, KimWipes | Kimberly-Clark | 34155 | Lab tissue wipers |
| Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished | Zeiss | Discontinued | Injection microscope |
| Methyl-4-hydroxybenzoate | Millipore-Sigma | H36471KG | Component of apple juice plates |
| Microinjector, FemtoJet4x | Eppendorf | 5253000025 | Microinjector |
| Micro loader tips, epT.I.P.S. 20 μL | Eppendorf | 5242956003 | For loading microneedles |
| Micromanipulator and injection stage with x,y,z dials for needle adjustment | Bernard Instruments, Inc (Houston, TX) | Custom | For performing microinjections |
| Micropipette puller, Model P-97, Flaming/Brown | Sutter Instruments | P97 | For pulling capillary tubes to make microneedles |
| Microscope cover glass 24×50-1.5 | Fisher Scientific | 12544E | For mounting embryos |
| Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm | Fisher Scientific | 22037081 | For mounting embryos for injection |
| n-Heptane | Fisher Scientific | H3601 | Component of embryo glue |
| Objective, 10x | Zeiss | Discontinued | 10x objective for injection microscope |
| Objective, C-Apochromat 40x/1,2 W Korr. FCS | Zeiss | 4217679971711 | 40x water objective for confocal |
| Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm) | Zeiss | 4208529871000 | 25x mixed immersion objective for confocal |
| Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA | Zeiss | 4207829900799 | 63x oil objective for confocal |
| Paintbrush, Robert Simmons Expression E85 Pointed Round size 2 | Daler-Rowney | 038372016954 | For transferring embryos |
| Paper towels, Kleenex C-fold paper towels, white | Kimberly-Clark | 884266344845 | For blotting cell strainer |
| Pasteur pipette, 5 3/4 in | Fisher Scientific | 1367820A | For covering embryos with oil |
| Petri dish, glass, 100 x 20 mm | Corning | 3160102 | For humid incubation chamber |
| Petri dish, plastic, 60 x 15 mm | VWR | 25384092 | For apple juice plates |
| Pipette, Eppendorf Reference 0.5-10 μL | Eppendorf | 2231000604 | For loading the microneedle |
| Pipette tip, xTIP4 250 μL | Biotix | 63300006 | For adding embryo glue to coverslip |
| Razor blade | VWR | 55411050 | For cutting agar wedge, tape, pipette tips |
| Rhodamine-conjugated globular actin, human platelet (non-muscle; 4×10 μg) | Cytoskeleton, Inc. | APHR-A | G-actin^Red |
| Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps | Fisher Scientific | 033377 | For making embryo glue |
| Screw top jar, 16 oz | Nalgene | 000194414195 | For desiccating embryos |
| Stage micrometer | Electron Microscopy Sciences | 602104PG | For calibrating volume of G-actin injection |
| Sucrose | Millipore-Sigma | 840971KG | Component of apple juice plates |
| Trizma base | Millipore-Sigma | T15031KG | Component of G buffer |
| Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz | Lesaffre Yeast Corporation | 117929157002 | Component of yeast paste |
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Tags
Keywords:
Intranuclear Actin RodsLive Heat Stressed Drosophila EmbryosMicroinjectionActin Stress ResponseActin Rod AssemblyMutant ScreenStress ConditionsEmbryo HandlingEmbryo PositioningEmbryo MountingG-actin RedApple Juice AgarDechorionationEmbryo TransferEmbryo AlignmentEmbryo GlueCoverslip Mounting
Citar este artículo
Biel, N., Figard, L., Sokac, A. M. Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos. J. Vis. Exp. (159), e61297, doi:10.3791/61297 (2020).