Name: 作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学
Uploaded: 2024-01-19T13:55:00
Description: Watch this Scientific Journal Video about Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research at JoVE.com

我们要感谢 Jennifer A. Zallen 博士提供 Sqh-GFP 果蝇 品系并为该技术的初始开发提供支持，并感谢 Francois Schweisguth 博士提供 Notch-GFP 果蝇 品系。这项工作得到了中国国家自然科学基金（32270809）对H.H.Yu的资助，南方科技大学生命科学学院的慷慨资助和人员支持，以及深圳市科学技术创新委员会/JCYJ20200109140201722对Y. Yan的资助。

实验是按照南方科技大学生命科学学院的指导和批准进行的。使用的生物体是黑腹果蝇，基因型是Notch-Knockin-GFP（X染色体）和Sqh-sqh-GFP（染色体II），分别由Francois Schweisguth博士（巴斯德研究所）和Jennifer Zallen博士（斯隆凯特琳研究所）的实验室慷慨提供。虽然该协议主要侧重于抗体标记和实时成像方面，但有关果蝇胚胎收集和注射的更详细描述，请参阅已发表的报告15,1...

通过抗体介导的实时成像增强蛋白质可视化

<ol>
	<li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginnings+of+immunofluorescence.">The beginnings of immunofluorescence.</a> Journal of Immunology (Baltimore, Md). 87, 499-503 (1961).</li><li>Arthur, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+the+antibody.">Harnessing the power of the antibody.</a> The Lancet Respiratory Medicine. 4 (3), 181-182 (2016).</li><li>Im, K., Mareninov, S., Diaz, M. F. P., Yong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobanking,+methods+and+protocols.">Biobanking, methods and protocols.</a> Methods in Molecular Biology. 1897, 299-311 (2018).</li><li>Ragkousi, K., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+integrity+and+cell+division:+Concerted+cell+cycle+control.">Epithelial integrity and cell division: Concerted cell cycle control.</a> Cell Cycle. 17 (4), 399-400 (2018).</li><li>Herszterg, S., Leibfried, A., Bosveld, F., Martin, C., Bellaiche, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+the+dividing+cell+and+its+neighbors+regulates+adherens+junction+formation+during+cytokinesis+in+epithelial+tissue.">Interplay between the dividing cell and its neighbors regulates adherens junction formation during cytokinesis in epithelial tissue.</a> Developmental Cell. 24 (3), 256-270 (2013).</li><li>Shimomura, O., Johnson, F. H., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction,+purification+and+properties+of+aequorin,+a+bioluminescent+protein+from+the+luminous+hydromedusan,+aequorea.">Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, aequorea.</a> Journal of Cellular and Comparative Physiology. 59 (3), 223-239 (1962).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263 (5148), 802-805 (1994).</li><li>Boshart, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+very+strong+enhancer+is+located+upstream+of+an+immediate+early+gene+of+human+cytomegalovirus.">A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.</a> Cell. 41 (2), 521-530 (1985).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Rodriguez, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+and+glowing+toolbox+of+fluorescent+and+photoactive+proteins.">The growing and glowing toolbox of fluorescent and photoactive proteins.</a> Trends in Biochemical Sciences. 42 (2), 111-129 (2016).</li><li>Berg, E. A., Fishman, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+antibodies+using+N+-Hydroxysuccinimide+(NHS)-fluorescein.">Labeling antibodies using N -Hydroxysuccinimide (NHS)-fluorescein.</a> Cold Spring Harbor Protocols. 2019 (3), (2019).</li><li>Saerens, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+universal+VHH+framework+to+graft+non-canonical+antigen-binding+loops+of+camel+single-domain+antibodies.">Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies.</a> Journal of Molecular Biology. 352 (3), 597-607 (2005).</li><li>Loncar, D., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+membrane+formation+during+the+cellularization+of+the+syncytial+blastoderm+of+Drosophila.">Cell membrane formation during the cellularization of the syncytial blastoderm of Drosophila.</a> Proceedings of the National Academy of Sciences. 92 (6), 2199-2203 (1995).</li><li>Lye, C. M., Naylor, H. W., Sanson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localisations+of+the+CPTI+collection+of+YFP-tagged+proteins+in+Drosophila+embryos.">Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos.</a> Development. 141 (20), 4006-4017 (2014).</li><li>Iordanou, E., Chandran, R. R., Blackstone, N., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+interference+by+dsRNA+injection+into+Drosophila+embryos.">RNAi interference by dsRNA injection into Drosophila embryos.</a> Journal of Visualized Experiments. 50, e2477 (2011).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+shape+change+in+living+Drosophila+embryos.">Imaging cell shape change in living Drosophila embryos.</a> Journal of Visualized Experiments. 49, e2503 (2011).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+development+of+Drosophila+melanogaster.">The embryonic development of Drosophila melanogaster.</a> , 9-102 (1997).</li><li>Ho, D. M., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch-mediated+proliferation+circuitry.">The Notch-mediated proliferation circuitry.</a> Current Topics in Developmental Biology. 116, 17-33 (2016).</li><li>Kopan, R., Ilagan, X. G., Ma, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+canonical+Notch+signaling+pathway:+unfolding+the+activation+mechanism.">The canonical Notch signaling pathway: unfolding the activation mechanism.</a> Cell. 137 (2), 216-233 (2009).</li><li>Fehon, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+between+the+protein+products+of+the+neurogenic+loci+Notch+and+Delta,+two+EGF-homologous+genes+in+Drosophila.">Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.</a> Cell. 61 (3), 523-534 (1990).</li><li>Struhl, G., Greenwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+is+required+for+activity+and+nuclear+access+of+Notch+in+Drosophila.">Presenilin is required for activity and nuclear access of Notch in Drosophila.</a> Nature. 398 (6727), 522-525 (1999).</li><li>Henrique, D., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Notch+signaling:+a+simple+logic+deployed+in+time+and+space.">Mechanisms of Notch signaling: a simple logic deployed in time and space.</a> Development. 146 (3), dev172148 (2019).</li><li>Trylinski, M., Mazouni, K., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-lineage+fate+decisions+involve+of+notch+receptors+basal+to+the+midbody+in+drosophila+sensory+organ+precursor+cells.">Intra-lineage fate decisions involve of notch receptors basal to the midbody in drosophila sensory organ precursor cells.</a> Current Biology. 27 (15), 2239.e3-2247.e3 (2017).</li><li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinases+and+phosphatases:+The+Yin+and+Yang+of+protein+phosphorylation+and+signaling.">Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling.</a> Cell. 80 (2), 225-236 (1995).</li><li>Glenney, J. R., Zokas, L., Kamps, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+phosphotyrosine.">Monoclonal antibodies to phosphotyrosine.</a> Journal of Immunological Methods. 109 (2), 277-285 (1988).</li><li>Hunter, T., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+induces+rapid+tyrosine+phosphorylation+of+proteins+in+A431+human+tumor+cells.">Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells.</a> Cell. 24 (3), 741-752 (1981).</li><li>Yu, H. H., Zallen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abl+and+Canoe/Afadin+mediate+mechanotransduction+at+tricellular+junctions.">Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions.</a> Science. 370 (6520), eaba5528 (2020).</li><li>Martin, A. C., Kaschube, M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+contractions+of+an+actin&#8211;myosin+network+drive+apical+constriction.">Pulsed contractions of an actin&#8211;myosin network drive apical constriction.</a> Nature. 457 (7228), 495-499 (2009).</li></ol>

该介绍的程序概述了使用定制抗体进行荧光标记并随后注射到早期 果蝇 胚胎中的专用方法。该技术有助于实时可视化少量存在的蛋白质或翻译后修饰，这些蛋白质或翻译后修饰通常难以通过传统的GFP/mCherry标记方法观察到。
在扩展该方法以在野生型和突变型胚胎之间进行定量比较时应谨慎行事。虽然在免疫荧光中，对照组和实验组之间的一抗和二抗浓度可以保持相同?...

免疫荧光是现代细胞生物学的一项基石技术，最初由 Albert Coons 开发，它能够检测其天然细胞区室的分子并表征亚细胞器或机器的分子组成1。结合基因操作，免疫荧光有助于建立现在广为接受的概念，即蛋白质定位对其功能至关重要2。除了特异性一抗和明亮的荧光染料外，该技术的成功还依赖于称为固定和透化的初步过程，该过程保留了细胞形态，固定了抗原，并增加了抗体进入细胞内区室的可及性。不可避免地，固定和透化过程会杀死细胞并终止所有生物过程3.因此，免疫荧光仅提供蛋白质生命旅程的快照。然而，许多生物过程（如细胞迁移和分裂）本质上是动态的，需要以时空分辨的方式研究蛋白质行为4,5。
为了研究生物体中的蛋白质动力学，已经开发了基于基因编码荧光蛋白（如绿色荧光蛋白 （GFP）6 和高速共聚焦显微镜）的实时成像方法。简而言之，目标蛋白可以通过基因操作与GFP7融合，然后从病毒或酵母启动子（如巨细胞病毒（CMV）8 或上游激活序列（UAS）9）异位表达。由于GFP本质上是自发荧光的，因此不需要荧光团偶联抗体来揭示靶蛋白的定位，从而绕过了固定或透化的初步过程的必要性。在过去的二十年中，已经开发了跨越整个波长光谱的荧光标签10，可以同时对多种靶蛋白进行多色实时成像。然而，与化学工程荧光染料（如AlexaFluor或ATTO）相比，这些基因编码的荧光蛋白的自发荧光在内源性启动子表达时相对较弱且不稳定，特别是在较长时间尺度的实时成像期间10。虽然这种不足可以通过过度表达荧光标记的靶蛋白来缓解，但许多具有酶活性的蛋白（如激酶和磷酸酶）如果不在生理水平上表达，会严重破坏正常的生物过程。
该协议提出了一种在实时图像设置中实现基于光稳定抗体的靶标照明的方法，基本上允许免疫荧光，而无需固定或透化过程（图1）。通过简单的基于 NHS 的伯胺反应11，可以将荧光染料（如 AlexaFluor 488 或 594）与基本上任何一抗或 GFP/HA/Myc 纳米抗体12 偶联。利用所有 果蝇 胚胎细胞在第 13 阶段共享共同细胞质的发育特征，可以在注射染料偶联抗体后实现整个胚胎的抗原结合和照明。随着 果蝇 和其他模型系统14中可用的内源性标记蛋白库的扩展，该方法可以通过揭示活组织中低丰度荧光标记蛋白和其他非荧光标记（HA/Myc标记）蛋白的动力学来潜在地扩大这些文库的应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech</td>
			<td>&#160;A620014&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Antibody Labeling Kit</td>
			<td>Invitrogen&#160;</td>
			<td>A20185</td>
			<td>Purification column from step 1.6 is included in this kit</td>
		</tr>
		<tr>
			<td>Biological Microscope</td>
			<td>SOPTOP</td>
			<td>EX20</td>
			<td>Eyepiece lens: PL 10X/20. Objective lens: 10x/0.25</td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox&#174;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>FALCON</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccation chamber&#160;</td>
			<td>LOCK&#38;LOCK</td>
			<td>HSM8200</td>
			<td>320ml</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Mshot</td>
			<td>MZ62</td>
			<td>Eyepiece lens: WF10X/22mm.</td>
		</tr>
		<tr>
			<td>Double-sided Tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Super Tweezer</td>
			<td>VETUS</td>
			<td>ST-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Cover Glasses: Rectangles</td>
			<td>Fisherbrand</td>
			<td>12-545F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Superfrost&#8482; Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>VETUS</td>
			<td>33A-SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 27</td>
			<td>Sigma-Aldrich</td>
			<td>H8773-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Sigma-Aldrich</td>
			<td>H2198-1L</td>
			<td>Heptane glue is made of double-sided tape immersed in heptane</td>
		</tr>
		<tr>
			<td>Dehydration reagent&#160;</td>
			<td>TOKAI</td>
			<td>1-7315-01</td>
			<td>Fill to 90% volume of the dessication chamber</td>
		</tr>
		<tr>
			<td>Manual Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>World Precision Instruments</td>
			<td>PUL-1000</td>
			<td>Procedure: step 1, Heat: 290, Force:300, Distance:1.00, Delay:50. 
			Step 2, Heat: 290, Force:300, Distance:2.21, Delay:50&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic picopump</td>
			<td>World Precision Instruments</td>
			<td>PV 830</td>
			<td>Eject: 20 psi;&#160; Range: 100ms; Duration: timed</td>
		</tr>
		<tr>
			<td>PY20</td>
			<td>Santa Cruz</td>
			<td>SC-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Square petri dishes</td>
			<td>Biosharp</td>
			<td>BS-100-SD</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP nanobody</td>
			<td>Chromotek</td>
			<td>gt</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing low-abundance proteins and post-translational modifications in living drosophila embryos via fluorescent antibody injection

使用 GFP（绿色荧光蛋白）和其他荧光标签对活细胞中的蛋白质进行可视化，极大地提高了对蛋白质定位、动力学和功能的理解。与免疫荧光相比，实时成像更准确地反映了蛋白质的定位，而不会因组织固定而产生潜在的伪影。重要的是，实时成像能够对蛋白质水平和定位进行定量和时间表征，这对于理解细胞运动或分裂等动态生物过程至关重要。然而，荧光标记方法的一个主要局限性是需要足够高的蛋白质表达水平才能实现成功的可视化。因此，无法检测到许多表达水平相对较低的内源性标记荧光蛋白。另一方面，使用病毒启动子的异位表达有时会导致蛋白质错定位或生理环境中的功能改变。为了解决这些局限性，提出了一种在活胚胎中利用高灵敏度抗体介导的蛋白质检测的方法，基本上无需组织固定即可进行免疫荧光。作为原理证明，在活胚胎中几乎无法检测到的内源性 GFP 标记的 Notch 受体在抗体注射后可以成功可视化。此外，这种方法适用于可视化活胚胎中的翻译后修饰 （PTM），允许检测早期胚胎发生过程中酪氨酸磷酸化模式的时间变化，并揭示顶膜下磷酸酪氨酸 （p-Tyr） 的新亚群。这种方法可以修改以适应其他蛋白质特异性、标签特异性或 PTM 特异性抗体，并且应该与其他适合注射的模式生物或细胞系兼容。该协议为低丰度蛋白质或PTM的实时成像开辟了新的可能性，而这些蛋白质或PTM以前很难使用传统的荧光标记方法进行检测。

Immunofluorescence is a cornerstone technique of modern cell biology originally developed by Albert Coons, which enables the detection of molecules at their native cellular compartments and characterization of the molecular compositions of subcellular organelles or machineries1. Coupled with genetic manipulations, immunofluorescence helps establish the now well-accepted concept that protein localization is essential for its function2. Aside from specific primary antibodies and bright fluorescent dyes, the success of this technique relies on a preliminary process named fixation and permeabilization, which preserves cellular morphologies, immobilizes antigens, and increases the accessibility of antibodies into intracellular compartments. Inevitably, the fixation and permeabilization process would kill cells and terminate all biological processes3. Therefore, immunofluorescence only provides snapshots of the life journey of proteins. However, many biological processes such as cell migration and divisions are dynamic in nature, requiring investigation of protein behaviors in a spatial-temporally resolved manner4,5.
To examine protein dynamics in living organisms, live imaging methods based on genetically encoded fluorescent proteins such as green fluorescent protein (GFP)6 and high-speed confocal microscopes have been developed. Briefly, the protein of interest can be genetically manipulated to be fused with GFP7, and then ectopically expressed from viral or yeast promoters such as cytomegalovirus (CMV)8 or upstream activation sequence (UAS)9. Because GFP is autofluorescent in nature, no fluorophore-coupled antibodies are required to reveal the localization of target proteins, which bypasses the necessity of preliminary processes of fixation or permeabilization. Over the last two decades, fluorescent tags spanning the whole spectrum of wavelength have been developed10, enabling multi-color live imaging of several target proteins at the same time. However, compared to chemically engineered fluorescent dyes such as AlexaFluor or ATTO, the autofluorescence of these genetically encoded fluorescent proteins is relatively weak and unstable when expressed from endogenous promoters, especially during live imaging over longer time scales10. While this shortfall can be mitigated by over-expressing fluorescently tagged target proteins, many with enzymatic activities such as kinases and phosphatases severely disrupt normal biological processes if not expressed at physiological levels.
This protocol presents a method that enables photostable antibody-based target illumination in a live image setup, essentially allowing immunofluorescence without the process of fixation or permeabilization (Figure 1). Through a simple NHS-based primary amine reaction11, one can conjugate fluorescent dyes such as AlexaFluor 488 or 594 with essentially any primary antibody or GFP/HA/Myc nanobody12. Taking advantage of a developmental feature that all Drosophila embryonic cells share a common cytoplasm during the syncytium stage13, one can achieve antigen binding and illumination across entire embryos after the injection of dye-conjugated antibodies. With expanding libraries of endogenously tagged proteins available in Drosophila and other model systems14, this method can potentially broaden applications of these libraries by revealing dynamics of low-abundance fluorescently tagged proteins and other non-fluorescently tagged (HA/Myc-tagged) proteins in living tissues.

作者聚焦：揭示动物形态发生研究中机械和生化信号的动力学 

通过荧光抗体注射观察活果蝇胚胎中的低丰度蛋白质和翻译后修饰

We aim to understand how mechanical and biochemical signals correlate animal morphogenesis, including the detection and the response to forces by cells and molecules. More molecules have been discovered to be mechanosensitive, functioning differently under varying levels of tension. However, the physiological context and the function of these force-sensitive events remain largely unknown.Due to the dynamic nature of force-sensitive events, our field largely relies on high-speed fluorescence imaging and time-lapse recording to capture molecular and cellular behaviors. Many fluorescently-tagged proteins are not bright enough to be imaged, and it requires spatial-temporal resolution without significant bleach. On the other hand, traditional immunofluorescence methods can only capture a snapshot of molecular and cellular fixture after fixation.This method paves the way to dynamically track the localization of many low-abundance protein receptors of major signaling pathways, such as Notch, Wnt, and the BMP pathways. Eventually, in addition to the traditional linear signaling cascades, we can pinpoint and where the signaling events occur at the subcellular scales.

为了证明抗体注射方法相对于基于荧光标签的实时成像或免疫荧光的优势，提供了两个案例研究，以表征低丰度跨膜受体Notch的动态定位，以及一种称为酪氨酸磷酸化的翻译后修饰在活胚胎中。
Notch信号转导活性在胚胎发生和成体器官稳态过程中的细胞命运决定中起主要作用18,19。在被其配体 Delta/Jagged20 激活后，?...

Watch this Scientific Journal Video about Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research at JoVE.com

该协议描述了定制的基于抗体的荧光标记和注射到早期 果蝇 胚胎中，以实现使用传统GFP / mCherry标签方法难以检测的低丰度蛋白质或翻译后修饰的实时成像。

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

我们旨在了解机械和生化信号如何与动物形态发生相关联，包括细胞和分子对力的检测和响应。更多的分子被发现是机械敏感的，在不同程度的张力下功能不同。然而，这些力敏感事件的生理背景和功能在很大程度上仍然未知。由于力敏感事件的动态性质，我们的领域在很大程度上依赖于高速荧光成像和延时记录来捕捉分子和细胞行为。许多荧光标记的蛋白质不够亮，无法成像，并且需要时空分辨率而没有明显的漂白剂。另一方面，传统的免疫荧光方法只能在固定后捕获分子和细胞固定装置的快照。该方法为动态跟踪主要信号通路（如Notch、Wnt和BMP通路）的许多低丰度蛋白受体的定位铺平了道路。最终，除了传统的线性信号级联之外，我们还可以确定信号转导事件在亚细胞尺度上发生的位置。

visualizing-low-abundance-proteins-post-translational-modifications

Research

JoVE Journal

Developmental Biology

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.