帕特里克卡弗蒂是从博士后奖学金，加拿大多发性硬化症协会的支持。

第1部分：预实验设置。 <ol><li>提前一周，队友苍蝇产生幼虫，表达绿色荧光蛋白胶质特异性启动子的控制下。对于我们的实验中，我们的可视化与核本地化，在采用反极性（回购）子（3，4）的神经胶质细胞中的表达序列标签的GFP。 </li><li>准备盖玻片提前至少一天18毫米的圆形盖玻片浸泡10分钟，在1％多聚- L -赖氨酸的解决方案和空气干燥过夜。 </li><li>在实验前一天，浸泡70％乙醇过夜的组件在一个清洁Chamlide的磁文化室。 </li><li>实验当天，准备加入胎牛血清，青霉素，链霉素的解决方案，与胰岛素施耐德的昆虫媒介使用无菌技术，以10毫升培养基。工作浓度：1X小牛血清，100U/ml青霉素链霉素为0.1mg/ml;和0.2毫克/毫升（修改（5））在施耐德的昆虫媒介的胰岛素。 </li><li>允许的文化室，空气干细胞培养罩。从文化室配制的培养液冲洗洗掉残留的乙醇。 </li></ol>第2部分： 果蝇眼脑复杂的夹层。 <ol><li>选择从一侧飞瓶砸冰的冷冻培养基的培养皿上，几分钟第三龄徘徊幼虫。冷幼虫建议缓慢蠕动身体收缩，便于清扫。 </li><li>放置在培养基上一个Sylgard涂下解剖镜下菜下降的冰鲜幼虫。 </li><li>解剖显微镜下，用一双杜蒙罚款钳，牢牢把握幼虫从后部的方式之一，大约三分之一。等待，直到幼虫挤出前结束其口钩。抓住嘴用的镊子完全伸展后的第二对挂钩。解剖幼虫，慢慢拉向相反的方向钳的两对。眼脑的复杂，唾液腺，脂肪组织，和成虫组织一起，将拉远从幼虫体内。不要拉口挂钩太快，否则会撕裂眼脑的复杂。 </li><li>修剪了唾液腺，脂肪组织，并采用超细剪剪成虫光盘。两半球的大脑和眼睛光盘将被连接到腹神经索和嘴钩。 </li><li>将18毫米到载玻片盖玻片。留下一个挂过的幻灯片的边缘，为了以后拿起盖玻片盖滑的边缘。新增的培养基下降到18毫米的盖玻片。转移到培养基中，通过抓钳嘴钩眼脑复杂。不要眼脑的复杂，直接掌握产钳或组织将被损坏。 </li><li>用细清扫剪刀，剪掉口眼脑的复杂和丢弃挂钩。实时成像的重要去除口钩口钩子将继续在文化造成的组织在显微镜移动介质合同。 </li><li>可视胶质细胞迁移眼内的成虫光盘，仔细切光秆，连接大脑和眼睛光盘的薄组织，并推搡或丢弃的大脑（见图1A）。要可视化的光纤秸秆内神经胶质细胞，离开大脑，视神经秆，眼睛成虫光盘完好无损（见图1E）。 </li><li>使用镊子拿起盖玻片下方周围组织的利益与永久性记号笔画一个圆圈。这个循环将有助于在显微镜定位在第4部分组织。 </li></ol>第3部分：安装在磁场文化室眼成虫光盘。 <ol><li>使用镊子，抓住挂在盖玻片边缘。盖玻片Chamlide磁腔的底板，在文化，而不会干扰组织。 </li><li>放置到主体Chamlide室硅胶O型圈。安装底板上的主体。 </li><li>慢慢加入培养液室。不要太快或组织的利益将受到干扰培养基添加。不要完全填补了腔使气体交换的掩护下。 </li><li>轻轻将无形Chamlide室顶部覆盖。 </li></ol>第4部分：可视胶质细胞迁移的眼睛光盘。 <ol><li>将共聚焦荧光显微镜舞台上的文化室。找到使用圆盖玻片作为一个低倍率的参考样本。 </li><li>使用40X镜头聚焦样本。允许组织盖玻片上解决。捕捉图像的眼睛光盘或光纤秸秆每15分钟3-4小时，10。该组织可能会抽搐和迁出的重点。手动对焦组织图像采集前将缓解次是问题。 </li></ol>第5部分：代表结果： 正确执行，我们的协议允许我们收集了一系列眼成虫光盘（图1屋宇署）迁移到视神经柄GFP标记的神经胶质细胞的图像。虽然60分钟的时间内实时成像足够的观察野生型眼成虫光盘内的变化，胶质细胞核的位置，在神经胶质细胞迁移所需的基因突变神经胶质核完全没有退出视神经柄（箭头图1 FH）。 我们有长达240分钟，然后观察培养组织的恶化时期培养的眼脑复合物。经过240分钟的时间点，我们开始观察周围的文化培养眼脑配合物（箭头图2 c）中等GFP阳性细胞。此外，绿色荧光蛋白会积聚在弥漫整个组织建议在组织的完整性崩溃点。 <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> 图1：在发展中的野生型和突变体的视觉系统的神经胶质细胞核GFP标记的实时成像。 A，E）的图像，使用微分干涉对比（DIC）的培养野生型和突变眼成虫光盘（ED）的显微镜。在野生型 ，出生于神经胶质细胞和视神经柄（OS）迁移到眼盘。大脑已被删除，以方便扁平化和成像眼成虫光盘。 BD）的荧光显微镜观察野生型的培养眼成虫光盘（一）为0，30和60分钟的时间点发现GFP标记神经胶质细胞核移植眼内盘。 FH）的突变眼脑复杂的荧光显微镜（e）在0，30和60分钟的时间点，展示了在视神经柄（箭头）的GFP标记神经胶质细胞核的拖延。大脑（BR）已经连接到光纤秆茎内的神经胶质细胞，以方便成像。 <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> 图2：实时成像GFP标记的胶质膜，在野生型眼成虫光盘。我们已经成功地培养了240分钟的时间眼脑的配合物。组织培养时间超过240分钟，开始打破。培养基周围的眼睛成虫光盘（ED）的，视柄（OS），0（A）和150分（b）和脑叶（BR），300分钟的时间点（C）相比，是免费的GFP阳性细胞，用箭头表示。

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</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, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., 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=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

在这个协议中，我们描述了进入眼内的成虫光盘使用Live显微镜观察神经胶质细胞迁移。在我们的野生型的例子（图1，AD），我们使用核GFP标记神经胶质细胞的运动超过一小时的过程中，观察的眼睛光盘。在胶质细胞迁移的需要在我们的实验室目前正在研究的一个候选基因的突变体，我们观察到，在一个小时内（图1 EH）的拖延胶质细胞的细胞核内的光纤秸秆。我们的策略，可以适应可视化?...

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		<th>Comment</th>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
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<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
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<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

脊椎动物和无脊椎动物的生物的神经胶质细胞迁移到最终目标地区，以ensheath，并支持相关的神经元。虽然最近已经取得了进展来形容发展蛹翼（1），研究神经胶质细胞的实时迁移<em&gt;果蝇</em&gt;胶质细胞迁移，通常涉及固定组织的考试。生活的游动细胞的微观分析提供了在整个迁徙过程中，包括确定率和生长方向的变化细胞行为能力的考查。配合使用基因工具，实时成像可以被用来确定在发展过程中特定基因的更精确的角色。以往的工作由Silies<em&gt;等。</em&gt;（2）描述来自光秆，一个发展中国家的眼睛和大脑的结构连接的神经胶质细胞迁移，进入眼内固定的组织成虫光盘。在这里，我们研究神经胶质细胞的实时迁移到一个协议大纲<em&gt;果蝇</em&gt;眼成虫光盘。我们采取了一个果蝇线的优势，遵循在野生型和突变体动物的神经胶质细胞的进展，对发展中的神经胶质细胞的GFP。

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

在这里，我们描述了一个协议，以研究胶质细胞迁移到发展果蝇眼睛使用现场的微观分析与GFP标记的神经胶质细胞配对。

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

live-imaging-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.7K 次观看.  University of British Columbia - UBC. 在这里，我们描述了一个协议，以研究胶质细胞迁移到发展果蝇眼睛使用现场的微观分析与GFP标记的神经胶质细胞配对。

视频: Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

科研

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动:Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

住在果蝇眼睛成虫光盘胶质细胞迁移成像

摘要

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从3龄果蝇幼虫的成虫光盘夹层

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料

一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇

实时成像果蝇胚胎血细胞迁移

影像生活细胞的形状变化果蝇胚胎

GFP标记的蛋白质在实时成像

在活细胞中蛋白质聚集的4D成像

凋亡细胞清除过程中实时成像

活细胞成像早期自噬活动:Omegasomes与超越

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像

住在果蝇眼睛成虫光盘胶质细胞迁移成像

摘要

探索更多视频

此视频中的章节

从3龄果蝇幼虫的成虫光盘夹层

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇

实时成像果蝇胚胎血细胞迁移

影像生活细胞的形状变化果蝇胚胎

GFP标记的蛋白质在实时成像

在活细胞中蛋白质聚集的4D成像

凋亡细胞清除过程中实时成像

活细胞成像早期自噬活动:Omegasomes与超越

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像

可视化的Live果蝇胶质细胞神经肌肉交界处，与荧光染料