ITSN，PI3K的C2β，并在本议定书中所使用的控制向量都可以从作者的请求后，仅用于非商业目的。作者要感谢张博士邓小平胡锦涛请提供意见和建立在O&#39;Bryan实验室附设协议中所用的试剂。卡乌是从基金会杰罗姆勒琼的资金支持。在O&#39;Bryan实验室的工作是由美国国立卫生研究院（HL090651），国防部（PR080428），圣Baldrick的基金会，基金会杰罗姆勒琼的赠款支持。

A.附设校准<ol><li> 选择一个荧光基团 ，有多种荧光团，如YFP和金星，以及附设的融合伙伴（见表1）工作。金星的氨基和羧基端的两端都能够形成一个复杂的，在37 ° C，而YFP的附设片段需要在30 ° C预孵化，以便荧光基团形成2。这在低温的潜伏期可能会改变一些细胞过程，并在选择的片段时，应考虑。融合金星蛋白质的羧基端向量Addgene <a href="http://www.addgene.org/pgvec1">（</a> http://www.addgene.org/pgvec1; seepBiFC VN173和pBiFC - VC155）伴随着额外的使用结构，作为对照， <a href="http://www.addgene.org/pgvec1?f=c&amp;identifier=22012&amp;atqx=BiFC&amp;cmd=findpl">例如，</a> pBiFC bJunVN173 pBiFC - bFosVC155 2。额外的载体，包括氨基端的金星向量（PFLAG VN173和PHA - VC155），可在下列<a href="http://people.pnhs.purdue.edu/%7Ehu1/">网站：http://people.pnhs.purdue.edu/〜</a> HU1 /。 </li><li> 标签蛋白的利益。附设片段融合的候选蛋白的氨基或羧基端两端。一些蛋白质，可能不会允许破坏蛋白质的功能，由于在两端进行标记。例如，Ras超家族的GTP酶的许多成员都在为此附设片段的羧基端，从而排除了附件修改的脂质。因此，重要的是有一些想法如何附设片段的附件可能会影响感兴趣的蛋白质的功能。如果不清楚如何标记的蛋白质会影响其功能的多种组合进行测试。此外附设片段，一种肽类连接器可包括增加零散的荧光基团和候选蛋白之间的灵活性。虽然多克隆位点（MCS），在附设载体编码的氨基酸短延伸，可提供足够的灵活性，RSIAT，KQKVMNH，和RPACKIPNDLKQKVMNH连接器已经成功地使用在 3,9附设实验</li><li> 确定转染条件。测试多个突变之前，应该进行一些控制实验。前两个附设组合应该尝试两个野生型蛋白是众所周知的互动和一个野生型和突变体不相互影响。使用这两种组合，DNA和转染时间的不同，应测试一个候选蛋白附设信号检测，以确定最佳条件。我们建议了一个6孔盘单井测试0.25ug，0.5ug，每个附设兴建1.0ug。一天后，通过荧光显微镜观察转染显示器的细胞，以确定信号发展的最佳时机。 <a href="http://www.addgene.org/pgvec1?f=c&amp;identifier=22012&amp;atqx=BiFC&amp;cmd=findpl">pBiFC bJunVN173</a>和pBiFC - bFosVC155 2构造附设有用的阳性对照和Addgene（见上文）。此外，应进行Western blot分析确认的构造平等的表达。荧光信号是两个野生型蛋白，但几乎没有信号之间的观察是观察野生型和突变蛋白之间，应选择条件。最后，最好是保持尽可能低蛋白的表达，以防止任何非特定的相互作用。 </li><li> 确定附设片段此外，如果改变了利益的蛋白质的本地化 。每个附设构建含有HA或标志抗原表位标记。免疫染色细胞transefected医管局或标志抗原表位标记以及良好的内源性蛋白（如果可能的话），以确定是否附设标签会影响利益的蛋白质本地化。 </li></ol> B.电镀和转染的细胞<ol><li> COS细胞（1.3x10 5）镀到玻璃底部Matek板和两个井，每个样品的6孔板。允许细胞一夜之间，收于37 ° C。也可用于更多的相关利益的候选蛋白的替代细胞类型。 </li><li>准备转染的DNA。我们通常使用的COS转染脂质体（Invitrogen公司）。然而，其他试剂可能更感兴趣的细胞株的适当。由于转染混合物将一个玻璃底菜和两个井6孔板之间的分割，使用适量的DNA占这个师。 （SF）的无血清DMEM培养液250uL稀释的DNA。添加CFP附设的DNA转染控制的总金额的1 / 5。附设量化，这是积极的只对CFP细胞将被分析为一个附设信号的存在。请注意，CFP光谱将与表1中的附设对一些重叠，因此​​，一个备用转控制也许需要。稀释在250uL SF DMEM（10uL Lipofectamine/1ug的DNA）的脂质体。混合DNA和脂质体稀释。在室温下孵育20分钟。 注：脂质体：DNA比例可以有所不同，取决于细胞类型。使用适当的转染法利益的细胞株。 </li><li> SF的DMEM用温水冲洗细胞2倍。新增SF的DMEM 2ML每个玻璃底板或一个6孔盘。 </li><li>每个转染混合物均匀地分割之间的一个玻璃底菜和两个井6以及菜。 </li><li>在37 ° C培养细胞为5小时。 </li><li>删除转媒体和完整的媒体取代（DMEM培养液10％胎牛血清）。 </li><li>隔夜孵育细胞在37 ° C孵化以下转的长度会有所不同，取决于利益的蛋白质表达水平。长时间的潜伏期，可能会导致非特异性相互作用，所以这一步将需要凭经验确定。 </li></ol> C.制备细胞成像<ol><li>一个啶，以确保阳性对照荧光显微镜下细胞的初步研究。如果不是，它可能是必要的，以便在37℃直至信号观察细胞额外的时间。 </li><li>冲洗细胞，用PBS液（pH 7.4）的3倍。在玻璃底培养皿的细胞增加了2％多聚甲醛（pH值7.4）。修正为10分钟在冰上的细胞。冲洗细胞，用PBS液（pH 7.4）的3倍。商店细胞在4 ° C，用1ml PBS（pH 7.4）中所涵盖的。细胞没有固定的成像，但一旦附设的片段，一个完整的荧光基团，它是不可逆转的3个动态的相互作用的分析，从而防止改革。另外，请记住，不固定的细胞会继续发展的信号。在6孔培养皿的裂解的细胞和准备免疫印迹分析裂解液。重要的是，所有细胞都在同一时间的准备，使裂解细胞成像细胞的代表。 </li></ol> D.成像细胞<ol><li>将计算每个细胞的荧光强度。确保单个细胞的图像。 </li><li> CFP资格被列入作为转染控制和唯一的CFP阳性细胞附设信号分析选择。我们假设，如果单元格与CFP转，也转与附设构造。这种方法可以确保成像细胞缺乏附设信号由于缺乏蛋白质的利益，而不是由于缺乏一个或两个单元格中的的附设表达结构之间的相互作用是否定的。 </li><li>我们使用的Zeiss LSM 510共聚焦显微镜观察细胞成像。使用这种显微镜时，重要的是要保持变焦，针孔，检测器增益，放大器的失调，帧大小，扫描速度，扫描平均，和激光功率一致。使用任何成像系统时，重要的是保持设置不变，使荧光样本之间的比较。此外，当量化荧光是很重要的像素都不会饱和。 </li></ol> E.定量荧光<ol><li>使用任何图像处理软件，可量化的荧光。我们利用ImageJ，这是从美国国立卫生研究院（http://rsb.info.nih.gov/ij/）免费提供。在ImageJ打开图像文件。 AnalyzeSet​​测量。检查区域的框和测量中的平均灰度值。 </li><li>使用的“自由之手的选择”工具，绘制一个围绕在CFP通道的整个细胞边缘的轮廓。 </li><li>这个纲要留在地方，转移到YFP的通道。 AnalyzeMeasure。 平均灰度值除以像素数（即平均每区的细胞荧光强度）的选择中的所有像素的灰度值的总和。 </li><li>对于每一个图像，画一个圈在一个地区，不包含细胞YFP的通道。采取这一领域作为背景的​​测量。从每幅图像中减去背景。 </li><li>平均的平均灰度值减去背景一个样本成像的所有细胞。这将是人口细胞的平均荧光强度。我们建议约60以上三个实验的细胞进行量化。 </li></ol> F.代表性的结果： 我们的实验室专注于多域的脚手架蛋白，与众多的蛋白质相互作用来调节多种生化和信号转导通路10,11,12,13,14 intersectin（ITSN ）。 ITSN包含两个Eps15（EH）的同源域，螺旋盘绕地区，和五个Src同源3（SH3）域。 ITSN不再亚型也包含后海湾干线的同源性（DH）和pleckstrin同源性（PH值）域的音乐会，作为一个鸟嘌呤核苷酸交换因子Cdc42 的 15 。这种模块化结构，促进蛋白质：蛋白质相互作用，使得ITSN附设实验的理想人选。 ITSN的亚细胞定位，可以改变其约束力的合作伙伴，并因此改变受ITSN（unpublis监管的途径HED数据）。最近，我们实验室已经证明，ITSN调节通过一种新型的Ⅱ类PI3K的PI3K的C2β11调节神经元的存活。丰富的PI3K的C2β域的氨基末端包含两个ITSN的SH3结构域的结合位点。我们使用ITSN和PI3K的C2β截断突变体免疫共沉淀表明，ITSN的SH3A和SH3C域与PI3K的C2β氨基末端区域互动。接下来，我们使用附设的可视化这一复杂的亚细胞定位。 ITSN是金星（PFLAG VN173）和PI3K的C2β结构的氨基端融合金星（PHA - VC155）羧基端融合。另一个阴性对照，是一种非特异性的肽融合到PHA - VC155。 VN - ITSN和VC - PI3K -C2β形成了一个断句的分布（图2A，上面板）一个附设的复杂。 PI3K的C2β扰乱ITSN和PI3K -C2β共沉淀丰富的临域的突变减少附设信号（图2A，低板）11 。这附设信号ITSN和两个PI3K的C2β蛋白质之间的差异是由于蛋白表达的差异（图2B）。 <img src="/files/ftp_upload/2643/2643fig1.jpg" alt="图1" /> 图1附设一个荧光团（在这种情况下，金星），分裂成氨基酸（越南） -羧基（VC）的终端两端。这些目标的融合，两种蛋白质的利益。当两种蛋白相互作用，VN和VC片段重组和互动的网站的荧光荧光重新关联。附设是一个特定的蛋白质片段互补，用来衡量蛋白质的测定（PCA）的例子：， 蛋白质相互作用5。 <img src="/files/ftp_upload/2643/2643fig2.jpg" alt="图2" /> 图2。ITSN和PI3K的C2β形成一个附设复杂。 A. VN -标签ITSN的VC标签C3βPI3K的WT或富含脯氨酸域合作tranfected突变（PI3K的C2β- PA）。 ITSN和WT PI3K的C2β形成一个复杂的（绿色）。 CFP（红色）被用来作为转染控制B.一个免疫印迹证明平等的构造表达。医管局标签的VC -标签的结构。 <img src="/files/ftp_upload/2643/2643table1.jpg" alt="表1" /> 表1，有多个向量附设兼容。 YN155：YFP的1 - 155aa; YC155：YFP的155 - 238aa; YN173：YFP的1 - 172aa; YC173，YFP的173 - 238aa; VN155：金星1 - 154aa; VC155：金星155-238; VN173： 1 - 172aa的金星; VC173：金星173 - 238aa; CN155 CFP资格1 - 154aa; CC155 CFP 155 - 238aa，GN173：绿色荧光蛋白1 - 172aa，CitN155：黄水晶1 - 155aa，CitC155：155 - 238aa CitN173 1 - 172aa：黄水晶，茶晶，CitC173：黄水晶173 - 238aa，CerN173：1 - 172aa蔚蓝2,3,9。

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附设是优秀的可视化蛋白质的方法：在整个细胞中的蛋白质相互作用，并确定这些复合物的亚细胞定位。附设的优势，只有相互作用的蛋白质荧光，相互作用的瞬态稳定，成像数据的后处理是最小的。这种方法的缺点是复杂的荧光团的荧光和不可逆转的成熟时间。在一些应用程序可以使用这个不可逆转的优势。例如，一个可使用附设复杂的酶，酶的活化剂，导致构激活。然后确定哪些蛋白质招募?...

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-013</td>
<td>&nbsp;</td>
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<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>35-011-CV</td>
<td>&nbsp;</td>
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<td>Glass Bottom Microwell dishes</td>
<td>&nbsp;</td>
<td>Matek</td>
<td>P35G-1.5-14C</td>
<td>&nbsp;</td>
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<td>6-well dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3846</td>
<td>&nbsp;</td>
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<td>Lipofectamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18324020</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-031-CV</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>LSM510 META</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

bimolecular fluorescence complementation

定义必须了解复杂的输出信号复合物的亚细胞分布。 常规方法，如免疫复合物的空间定位的信息不提供。相比之下，附设显示器的相互作用和细胞内的蛋白质复合物的条块分割。在此方法中，fluororescent蛋白质氨基和羧基端的非荧光片段，这两种蛋白质的利益，然后融合到分成。重组的荧光（图1）1,2的蛋白质结果的相互作用。附设的一个限制是，一旦重组分散的荧光基团是复杂的，是不可逆转的 3 。这种限制是在检测的瞬态或弱相互作用是有利的，但排除了复杂的动态的动力学分析。另外一个需要注意的是，重组后的flourophore需要30分钟，以成熟和荧光，再次解除观察实时交互4。附设的蛋白质片段互补法（PCA）的记者的蛋白质，如绿色荧光蛋白变异（附设），二氢叶酸还原酶，B -内酰胺酶和荧光素酶蛋白质员工具体的例子： 蛋白质相互作用5,6。替代的方法来研究蛋白质在细胞中的蛋白质相互作用，包括荧光共定位和福斯特共振能量转移（FRET）7 。合作的定位，两种蛋白质单独标签可以直接与间接免疫荧光基团或。然而，这种做法导致高背景的非相互作用的蛋白质，使其难以解释的合作本地化数据。此外，由于共聚焦显微镜分辨率的限制，两种蛋白质，可能会出现，而不必进行交互合作本地化。随着附设，荧光观察时，两种蛋白相互作用的利益。 FRET的是另一种优秀的方法研究蛋白质：蛋白质相互作用，但可以在技术上具有挑战性。 FRET实验要求供体和受体类似的亮度和在细胞内的化学计量。此外，必须考虑到受体通道，反之亦然通过捐助为出血。附设有不同的FRET，荧光图像数据的小背景，小的后处理，不需要很高的过度表达，并可以检测弱或短暂的相互作用。生物发光共振能量转移（BRET）是一个类似的担忧，除了捐助是一种酶（如荧光素酶）催化底物，从而令人兴奋的一个受体，成为发光的方法。 BRET没有流血通过技术问题和高背景荧光，但没有能力提供的空间信息，由于缺乏基板本地化的8个具体的车厢。总体而言，附设是一个可视化的蛋白质复合体的亚细胞定位要进入隔离的信号洞察力的优秀方法。

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation at JoVE.com

Bimolecular Fluorescence Complementation

在确定的时空调控细胞信号蛋白质的亚细胞定位是很重要的。在这里，我们描述了一个简单的方法监测在细胞内的蛋白质的空间相互作用的双分子荧光互补（附设）。

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Research

JoVE Journal

Biology

27.7K 次观看.  University of Illinois at Chicago. 在确定的时空调控细胞信号蛋白质的亚细胞定位是很重要的。在这里，我们描述了一个简单的方法监测在细胞内的蛋白质的空间相互作用的双分子荧光互补（附设）。

视频: Bimolecular Fluorescence Complementation

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

检查荧光模式Cryosectioning酵母社区

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

全基因组蛋白质相互作用通过蛋白质片段互补法（PCA）的活细胞筛选

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...