This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.

1.细胞培养<ol><li>使用端粒酶逆转录酶（hTERT）永生视网膜色素上皮细胞（hTERT的-RPE1;这里称为RPE1）。 注：RPE1细胞是那些通常用于研究中心粒装配和ciliogenesis近二倍体，未转化的人细胞。这些细胞遵循正常的中心粒复制周期协调与调控细胞周期。 </li><li>通道RPE1细胞的近汇合百毫米细胞培养皿以1：5稀释的原始培养物进含有10ml的新鲜100mm皿的DME / F-12（1：1）培养基补充有10％胎牛血清（FBS），100U / ml青霉素G和100微克/ ml链霉素（完全培养基被称作DME / F-12培养基）。在5％CO 2存在下生长的细胞在37℃。 <ol><li>孵育12毫米圆形盖玻片在50毫升的1N HCl的带盖的玻璃烧杯中，O / N不摇动，在60℃下在水浴或培养箱。 </li><li>自动对焦之三废弃的酸溶液，孵育15分钟，偶尔晃动洗涤盖玻片在100ml蒸馏水洗涤三次。在70％的乙醇和95％的乙醇同样地重复洗涤。 </li><li>通过单独地散布出来以实验室印迹纸在生物安全柜，随后灭菌用UV辐射60分钟，干燥该盖玻片。 </li></ol></li><li>将2-4干盖玻片35毫米的细胞培养皿。稀释纤连蛋白的溶液或纤连蛋白样工程聚合物（原料浓度为1毫克/毫升），以25微克/毫升细胞培养PBS中的浓度。 <ol><li>放置80-100微升的稀释液到每个盖玻片并孵育60分钟以涂覆的盖玻片的顶侧。洗盖玻片三次用PBS加入完全培养基之前。 </li></ol></li></ol> 2.生长的细胞和细胞治疗与蛋白酶体抑制剂<ol><li>通过2×10 5异步成长包含ING RPE1细胞中的每个35mm培养皿盖玻片和成长的细胞在2毫升完全培养基。更换培养液，每24小时用新鲜预热完全培养基。 <ol><li>来分析测试蛋白质的水平中心体蛋白酶体抑制的效果（这里VDAC3或γ微管蛋白），生长细胞两张35毫米的菜肴44小时。替换完全培养基的培养基中，在5微米或0.05％的最终浓度分别添加任MG115或DMSO作为对照的溶剂。与此同时，4小时添加BrdU的给细胞以40μM的终浓度，并培育细胞。 </li><li>转移在24孔板每个盖玻片。加入500微升冷却的甲醇到每个孔中并且孵育所述板在-20℃下10分钟以固定在盖玻片的细胞。立即清洗盖玻片三次，用500μL洗涤缓冲液（1×PBS中含0.5mM的MgCl 2和0.05％的Triton-X 100）。 </li></ol></li><li>收获残留细胞从35毫米培养皿，并离心将细胞在1000×g离心5分钟。使用细胞沉淀来分析由蛋白质印迹（步骤6）的总蛋白。 </li></ol> 3. BrdU的脉冲和大通分析来分析不同细胞周期蛋白水平<ol><li>为了分析蛋白质（此处VDAC3，SAS6或Cep135）的电平的变化，在中心体在细胞周期的不同阶段，在含有盖玻片为44小时2张35毫米培养皿生长的细胞。替换用含有40μM的BrdU完全培养基培养基孵育的细胞在细胞培养孵化4小时。 </li><li>来自一个培养皿，盖玻片转移到一个24孔板中步骤2.1.2描述以固定用冷却的甲醇的细胞。 </li><li>在4小时的BrdU脉冲之后，除去含有的BrdU的培养基，用PBS洗一次细胞，并一次用完全培养基，在不存在的BrdU不同的时间（通常是另一个4小时加入新鲜培养基，再生长的细胞对于如在步骤2.1.2中所述的固定的细胞前RPE1细胞）。 注：对于RPE1细胞，大多数BrdU阳性细胞都在后期S或G2期为4小时追。这必须独立地对每个细胞类型来确定。 </li></ol> 4.免疫染色<ol><li>孵育在200μl封闭缓冲液（2％BSA和0.1％的TritonX-100在1×PBS中），30分钟的盖玻片固定细胞。 <ol><li>孵育细胞与初级抗体的混合物（典型地一个凸起在兔和小鼠另一个凸起）稀释在封闭缓冲液O / N在4℃下在湿润的腔室中。 </li><li>为了使加湿室，把湿纸巾在底部空1000微升枪头盒一半。躺在机架表面封口膜的条带，并发现该抗体溶液的液滴（15-20微升）到封口膜为每个盖玻片培养。倒置盖玻片上的抗体溶液的液滴（使得细胞浸泡）和附近的尖端盒的盖子。 </li><li>再次反转盖玻片，并将其回回到24孔的菜。洗盖玻片，然后孵育它们在150微升二次抗体混合物（此处绿色荧光染料缀合的抗兔和红色荧光染料缀合的抗小鼠稀释于封闭缓冲液）处理1小时，在室温。洗盖玻片三次。 注：荧光团共轭次级抗体是光敏感的。保护过程中的步骤，从光样品4.1.3-5.1.2。 </li></ol></li><li>通过在-20℃下用500微升冷却的甲醇再次固定染色RPE1细胞10分钟，如在步骤2.1.2中所述制备用于抗BrdU染色。三次洗盖玻片。 注意：此固定期间在以下步骤中的酸水解过程固定感兴趣的蛋白质（多个）的初级和次级抗体标记。 <ol><li>孵育细胞在200μl的2N盐酸进行30分钟，在室温。中和用300微升的1M Tris-的Cl，pH值8的d。洗涤细胞用洗涤缓冲液三次。 </li><li>方框用200μl的封闭缓冲液30分钟，在RT下再细胞。 </li><li>孵育细胞与大鼠抗BrdU抗体（稀释于封闭缓冲液），45分钟，在37℃在湿润的腔室中。返回盖玻片回24孔皿中，把它们洗干净，然后孵育蓝色荧光染料缀合的抗大鼠第二抗体（稀释于150微升封闭缓冲液在24孔培养皿1小时，在室温。 </li></ol></li><li>洗盖玻片三次。发现含在玻璃显微镜载玻片抗淬灭剂的安装溶液的液滴（约3-6微升）。倒置盖玻片，与细胞侧朝下，到安装的解决方案。轻轻按压使用清洁的软组织幻灯片盖玻片擦去多余的液体。应用透明指甲油沿盖玻片的边缘，以密封其上的显微镜载玻片。 </li></ol> 5.免疫荧光图像Acquisition与分析<ol><li>使用100X计划的Apo油浸物镜（具有1.4的数值孔径），以获得BrdU阳性RPE1细胞的图像在环境温度下。 <ol><li>把幻灯片上的显微镜（见设备）附能够进行数字成像的相机。对于每一个荧光团，通过手动检查的实验的所有样品确定适当的曝光时间（一般为300-1,000毫秒）。取得的细胞的图像之前，手动确定适当的顶部和底部的焦平面沿Z轴（通常为0.2μm的步长）。获得不同的样品是在使用相同的曝光时间为沿着Z轴的不同的荧光团使用数字显微镜成像软件包单独盖玻片的图像。 </li></ol></li><li>执行解卷积（在这种情况下不使用邻算法）沿Z轴获得的所有的图像栈。 <ol><li>沿获得每个图像栈的总强度投影Z轴。 </li></ol></li><li>在细胞中，其中中心体接近（2中心体之间的距离小于2微米），画一个小方（通常每侧20-30像素）周围两中心体和标记所选区域。 <ol><li>画一个更大的方块（通常每侧24-35像素）围绕第一广场和纪念大广场的选定区域。 </li><li>得到的面积（A）和在每个框中的每个荧光团的总荧光强度（F），（S表示小框且L表示大框）。 </li><li>分析利用豪威尔等 29所描述的公式中的每个荧光背景校正的荧光强度：F = F 的S - [（F L - 五六）×A S /（A L - A S）]。 </li><li>通过计算其FL背景校正的荧光强度的比率获得的利息（此处VDAC3或SAS6）蛋白的标准化荧光强度uorophore根据与用于所选择的内标物（这里γ微管蛋白，SAS6或Cep135）的荧光团。 </li></ol></li><li>在分析单元，其中中心体完全分离的情况下，（2中心体之间的距离大于2微米），分别分析各中心体通过拉伸两个独立的盒套。画一个小方（通常每侧15-25像素）周围的每个中心体和标记所选区域。画一个更大的正方形（每侧20-30像素）周围的第一方阵，并标明所选区域。 <ol><li>得到的面积（A）和在每个框中的每个荧光团的总荧光强度（F）中，并计算每个荧光团的背景校正的荧光强度，分别对每个中心体，如在步骤5.3.3说明。 </li><li>结合各蛋白质的背景校正的荧光强度，从两中心体，以获得在该细胞蛋白质的总中心体水平。 </li><li>获取通过计算其总中心体强度的比率为内标物的总中心体强度归一化强度对感兴趣的蛋白质。 </li></ol></li><li>对于每个实验条件进行分析，至少15-25细胞并绘制图表中的电子表格。 </li></ol> 6.分析总蛋白使用免疫印迹<ol><li>重悬的细胞在放射免疫沉淀测定含有50mM的Tris-HCl pH为8，150 mM氯化钠，1％的Nonidet P-40，1％脱氧胆酸钠，0.1％十二烷基硫酸钠（SDS）（RIPA）缓冲液孵育在冰上10分钟以裂解细胞。 <ol><li>以10,000×g离心离心该混合物10分钟，分离沉淀级分的溶解产物，并用二辛可宁酸（BCA）测定试剂盒测定各裂解物的总蛋白浓度。 </li></ol></li><li>混合40微克裂解物与4倍的SDS-PAGE上样缓冲液（50毫摩尔Tris-CL，pH值6.8，2％SDS，10％甘油，100mM的二硫苏糖醇，0.1％维奇ophenol蓝色）。 <ol><li>煮沸样品10分钟，并运行变性SDS-PAGE上的12.5％的样品在200 V的恒定电压</li></ol></li><li>传送分离在SDS-PAGE上使用蛋白质印迹技术中的硝化纤维素膜的蛋白质（在90 V 1小时在寒冷的恒定电压）。 <ol><li>方框用3％无脂牛奶中的1×PBS的膜，然后孵育稀释的初级抗体溶液的膜（在此情况下的兔抗VDAC3，兔抗γ-tubulin和小鼠抗α微管蛋白）为1小时，在RT。 </li><li>洗膜用PBS-T缓冲三次（每次5分钟温育在摇动下）后（1×PBS和0.2％吐温-20），孵育该膜在近红外（IR）的荧光染料缀合的抗兔的混合物和红外荧光染料缀合的抗小鼠二抗1小时（稀释在PBS-T）的。 </li><li>洗膜三次，然后扫描该膜用红外f以分析频带luorescence成像系统适用于免疫印迹检测。 </li></ol></li></ol>

<ol>
	<li>Ganem, N. J., Godinho, S. A., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+linking+extra+centrosomes+to+chromosomal+instability.">A mechanism linking extra centrosomes to chromosomal instability.</a> Nature. 460, 278-282 (2009).</li><li>Kobayashi, T., Dynlacht, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+transition+from+centriole+to+basal+body.">Regulating the transition from centriole to basal body.</a> J. Cell Biol. 193, 435-444 (2011).</li><li>Azimzadeh, J., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+centriole.">Building the centriole.</a> Curr. Biol. 20, 816-825 (2010).</li><li>Nakazawa, Y., Hiraki, M., Kamiya, R., Hirono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAS-6+is+a+cartwheel+protein+that+establishes+the+9-fold+symmetry+of+the+centriole.">SAS-6 is a cartwheel protein that establishes the 9-fold symmetry of the centriole.</a> Curr. Biol. 17, 2169-2174 (2007).</li><li>Strnad, P., 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=Regulated+HsSAS-6+levels+ensure+formation+of+a+single+procentriole+per+centriole+during+the+centrosome+duplication+cycle.">Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle.</a> Dev. Cell. 13, 203-213 (2007).</li><li>Hilbert, 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=Caenorhabditis+elegans+centriolar+protein+SAS-6+forms+a+spiral+that+is+consistent+with+imparting+a+ninefold+symmetry.">Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 11373-11378 (2013).</li><li>Pike, A. N., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centriole+assembly+and+the+role+of+Mps1:+defensible+or+dispensable.">Centriole assembly and the role of Mps1: defensible or dispensable.</a> Cell Div. 6, 9 (2011).</li><li>Haren, L., Stearns, T., Luders, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plk1-dependent+recruitment+of+gamma-tubulin+complexes+to+mitotic+centrosomes+involves+multiple+PCM+components.">Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM components.</a> PLoS One. 4, e5976 (2009).</li><li>Andersen, J. S., 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=Proteomic+characterization+of+the+human+centrosome+by+protein+correlation+profiling.">Proteomic characterization of the human centrosome by protein correlation profiling.</a> Nature. 426, 570-574 (2003).</li><li>Keller, L. C., Romijn, E. P., Zamora, I., Yates, J. R., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+isolated+chlamydomonas+centrioles+reveals+orthologs+of+ciliary-disease+genes.">Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.</a> Curr. Biol. 15, 1090-1098 (2005).</li><li>Kumar, A., Rajendran, V., Sethumadhavan, R., Purohit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEP+proteins:+the+knights+of+centrosome+dynasty.">CEP proteins: the knights of centrosome dynasty.</a> Protoplasma. 250, 965-983 (2013).</li><li>Kanai, 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=Physical+and+functional+interaction+between+mortalin+and+Mps1+kinase.">Physical and functional interaction between mortalin and Mps1 kinase.</a> Genes Cells. 12, 797-810 (2007).</li><li>Jakobsen, L., 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=Novel+asymmetrically+localizing+components+of+human+centrosomes+identified+by+complementary+proteomics+methods.">Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods.</a> Embo. J. 30, 1520-1535 (2011).</li><li>Sang, L., 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=Mapping+the+NPHP-JBTS-MKS+protein+network+reveals+ciliopathy+disease+genes+and+pathways.">Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways.</a> Cell. 145, 513-528 (2011).</li><li>Dowdle, W. E., 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=Disruption+of+a+ciliary+B9+protein+complex+causes+Meckel+syndrome.">Disruption of a ciliary B9 protein complex causes Meckel syndrome.</a> Am. J. Hum. Genet. 89, 94-110 (2011).</li><li>Majumder, S., Slabodnick, M., Pike, A., Marquardt, J., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC3+regulates+centriole+assembly+by+targeting+Mps1+to+centrosomes.">VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes.</a> Cell Cycle. 11, 3666-3678 (2012).</li><li>Guderian, G., Westendorf, J., Uldschmid, A., Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plk4+trans-autophosphorylation+regulates+centriole+number+by+controlling+betaTrCP-mediated+degradation.">Plk4 trans-autophosphorylation regulates centriole number by controlling betaTrCP-mediated degradation.</a> J. Cell Sci. 123, 2163-2169 (2010).</li><li>Holland, A. J., 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+autoregulated+instability+of+Polo-like+kinase+4+limits+centrosome+duplication+to+once+per+cell+cycle.">The autoregulated instability of Polo-like kinase 4 limits centrosome duplication to once per cell cycle.</a> Genes Dev. 26, 2684-2689 (2012).</li><li>Kasbek, C., 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=Preventing+the+degradation+of+mps1+at+centrosomes+is+sufficient+to+cause+centrosome+reduplication+in+human+cells.">Preventing the degradation of mps1 at centrosomes is sufficient to cause centrosome reduplication in human cells.</a> Mol. Biol. Cell. 18, 4457-4469 (2007).</li><li>Kasbek, C., Yang, C. H., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antizyme+restrains+centrosome+amplification+by+regulating+the+accumulation+of+Mps1+at+centrosomes.">Antizyme restrains centrosome amplification by regulating the accumulation of Mps1 at centrosomes.</a> Mol. Biol. Cell. 21, 3878-3889 (2010).</li><li>Puklowski, 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+SCF-FBXW5+E3-ubiquitin+ligase+is+regulated+by+PLK4+and+targets+HsSAS-6+to+control+centrosome+duplication.">The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.</a> Nat. Cell Biol. 13, 1004-1009 (2011).</li><li>Spektor, A., Tsang, W. Y., Khoo, D., Dynlacht, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cep97+and+CP110+suppress+a+cilia+assembly+program.">Cep97 and CP110 suppress a cilia assembly program.</a> Cell. 130, 678-690 (2007).</li><li>Salisbury, J., Suino, K., Busby, R., Springett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrin-2+is+required+for+centriole+duplication+in+Mammalian+cells.">Centrin-2 is required for centriole duplication in Mammalian cells.</a> Curr. Biol. 12, 1287 (2002).</li><li>Lukasiewicz, K. B., 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=Control+of+centrin+stability+by+Aurora+A.">Control of centrin stability by Aurora A.</a> PLoS One. 6, e21291 (2011).</li><li>Zhao, J., Ren, Y., Jiang, Q., Feng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin+is+recruited+to+the+centrosome+in+response+to+inhibition+of+proteasomes.">Parkin is recruited to the centrosome in response to inhibition of proteasomes.</a> J. Cell Sci. 116, 4011-4019 (2003).</li><li>Korzeniewski, N., 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=Cullin+1+functions+as+a+centrosomal+suppressor+of+centriole+multiplication+by+regulating+polo-like+kinase+4+protein+levels.">Cullin 1 functions as a centrosomal suppressor of centriole multiplication by regulating polo-like kinase 4 protein levels.</a> Cancer Res. 69, 6668-6675 (2009).</li><li>Rohatgi, R., Milenkovic, L., Corcoran, R. B., Scott, 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=Hedgehog+signal+transduction+by+Smoothened:+pharmacologic+evidence+for+a+2-step+activation+process.">Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 3196-3201 (2009).</li><li>Majumder, S., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC3+and+Mps1+negatively+regulate+ciliogenesis.">VDAC3 and Mps1 negatively regulate ciliogenesis.</a> Cell Cycle. 12, 849-858 (2013).</li><li>Howell, B. J., Hoffman, D. B., Fang, G., Murray, A. W., Salmon, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Mad2+dynamics+at+kinetochores,+along+spindle+fibers,+and+at+spindle+poles+in+living+cells.">Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.</a> J. Cell Biol. 150, 1233-1250 (2000).</li><li>Meraldi, P., Nigg, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+cohesion+is+regulated+by+a+balance+of+kinase+and+phosphatase+activities.">Centrosome cohesion is regulated by a balance of kinase and phosphatase activities.</a> J. Cell Sci. 114, 3749-3757 (2001).</li><li>Zimmerman, W. C., Sillibourne, J., Rosa, J., Doxsey, 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=Mitosis-specific+anchoring+of+gamma+tubulin+complexes+by+pericentrin+controls+spindle+organization+and+mitotic+entry.">Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry.</a> Mol. Biol. Cell. 15, 3642-3657 (2004).</li><li>Lee, K., Rhee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLK1+phosphorylation+of+pericentrin+initiates+centrosome+maturation+at+the+onset+of+mitosis.">PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis.</a> J. Cell Biol. 195, 1093-1101 (2011).</li><li>Lin, Y. C., 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=Human+microcephaly+protein+CEP135+binds+to+hSAS-6+and+CPAP,+and+is+required+for+centriole+assembly.">Human microcephaly protein CEP135 binds to hSAS-6 and CPAP, and is required for centriole assembly.</a> Embo. J. 32, 1141-1154 (2013).</li><li>Lichtenstein, N., Geiger, B., Kam, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cytoskeletal+organization+by+digital+fluorescent+microscopy.">Quantitative analysis of cytoskeletal organization by digital fluorescent microscopy.</a> Cytometry A. 54, 8-18 (2003).</li><li>Keller, L. C., 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+architecture+of+the+centriole+proteome:+the+conserved+WD40+domain+protein+POC1+is+required+for+centriole+duplication+and+length+control.">Molecular architecture of the centriole proteome: the conserved WD40 domain protein POC1 is required for centriole duplication and length control.</a> Mol. Biol. Cell. 20, 1150-1166 (2009).</li><li>Venoux, 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=Poc1A+and+Poc1B+act+together+in+human+cells+to+ensure+centriole+integrity.">Poc1A and Poc1B act together in human cells to ensure centriole integrity.</a> J. Cell Sci. 126, 163-175 (2013).</li><li>Stevens, N. R., Roque, H., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSas-6+and+Ana2+coassemble+into+tubules+to+promote+centriole+duplication+and+engagement.">DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement.</a> Dev. Cell. 19, 913-919 (2010).</li><li>Machiels, B. 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=Detailed+analysis+of+cell+cycle+kinetics+upon+proteasome+inhibition.">Detailed analysis of cell cycle kinetics upon proteasome inhibition.</a> Cytometry. 28, 243-252 (1997).</li><li>Yang, C. H., Kasbek, C., Majumder, S., Yusof, A. M., Fisk, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mps1+phosphorylation+sites+regulate+the+function+of+centrin+2+in+centriole+assembly.">Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly.</a> Mol. Biol. Cell. 21, 4361-4372 (2010).</li><li>Tsou, M. F., 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=Polo+kinase+and+separase+regulate+the+mitotic+licensing+of+centriole+duplication+in+human+cells.">Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells.</a> Dev. Cell. 17, 344-354 (2009).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 2415-2420 (2008).</li><li>Buck, S. B., 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=Detection+of+S-phase+cell+cycle+progression+using+5-ethynyl-2'-deoxyuridine+incorporation+with+click+chemistry,+an+alternative+to+using+5-bromo-2'-deoxyuridine+antibodies.">Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.</a> Biotechniques. 44, 927-929 (2008).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> J. Cell Biol. 185, 1135-1148 (2009).</li></ol>

The authors declare that they have no competing financial interests.

在细胞生物学定量显微镜通常与活细胞成像试验，如荧光共振能量转移（FRET），荧光恢复漂白相关（FRAP） 等。然而，后有细胞生物学家的例子越来越多开发不同的定量检测显微镜对固定细胞近年来27,34-36。重要的是，了解中心体生物学的进步往往需要的蛋白质，其中心体池可能会受到不同的管制比其他池的中心体特异功能的理解。因此，我们开发了一个定量的IIF分析特异性分析的...

中心体由一对中心粒由pericentriolar材料（PCM）所包围的。为在哺乳动物细胞中主要的微管组织中心（MTOCs），中心体作为有丝分裂纺锤体的两极在分裂的细胞，从而有助于维持基因组完整性1。在静止期细胞（ 例如 ，在G0期）时，中心体，即母中心粒的两个中心粒中的一个，被转换成一个基体组装在主纤毛，感官细胞器从细胞表面2突出出来。一旦细胞重新进入细胞周期，原发性纤毛被拆卸并且每个中心粒指挥一个procentriole在其近端逐渐伸长，以形成一个成熟的中心粒3的组装。在S期的开始，一个侧手翻状结构，它提供了9倍对称性的中心粒形成每个现有中心粒的表面上，将成为各procentriole的基础。 SAS6吨帽子是必不可少的中心粒组件被招募来形成手翻4-6的中心。其他中心粒蛋白，然后组装到侧手翻在一个高度管制，近端远端的方式7。经过精确完成中心粒的复制，细胞聚集更多的pericentriolar材料由G2期8月底前建立两个功能中心体。在除了核心部件中心粒9-11，其它几种蛋白质，包括蛋白激酶，磷酸酶，蛋白伴侣，脚手架元件，膜相关蛋白和降解机械与中心粒，基体和PCM在细胞周期12-16的不同的时间相关联。它经常被注意的是，许多蛋白质​​的中心体的水平在时间上由中心体定位的机制和/或蛋白酶体降解在中心体调节。重要的是，在几个蛋白质如PLK4，MPS1，SAS6和CP110 a的中心体水平的波动吨的不同点的细胞周期似乎是至关重要的调节中心粒装配5,17-22，和在MPS1的防止这个中心体的降解的情况下会导致过量中心粒19的形成。另一方面，相对于细胞溶质池几种蛋白质的中心体部分较少不稳定。例如的siRNA介导的下调中金2（Cetn2）导致了在中心粒蛋白水平仅温和下降，尽管大大降低了整个细胞水平的23。因此，关键是要测量在中心体的变化中心体蛋白的水平，而不是评估其中心体特定的功能时，测定全细胞蛋白质的水平。 在这项研究中，我们开发了使用间接免疫荧光（IIF）量化蛋白质在中心体的相对水平的测定法。该测定被显影尤其来分析细胞，来自不同样品因此不能在同一时间被成像。这些样品可以是用不同的试剂（ 即 ，药物与对照）中，在不同的时间点收集经处理的细胞（ 即 ，脉搏与追），或者是在细胞周期的不同阶段。这个测定的原理在于测量对应于蛋白质在一个小区域和正常化针对同一该值对于另一种蛋白质，其水平所选择的实验条件下不发生变化的背景校正的荧光强度。在中心体生物学几项研究最近使用的各种定量显微技术，在这两种活的或固定细胞，以确定候选蛋白24-27的中心体特定的函数。类似测定法，本发明的技术还可以测量测试蛋白的背景校正的荧光强度。然而，在该测定法使用内标列入正常化将可能提供更多的准确性和信心在分析两种不同的样本是在两个不同的盖玻片。此外，除了在研究中心体蛋白质水平，用小的调整这种方法可以应用到的实验条件多样化或其它细胞位点。 在这里，我们结合定量检测显微镜用的BrdU脉冲追踪策略，比较不同的细胞周期阶段的细胞。代替使用标准的细胞周期停滞和释放技术，研究各种细胞周期的时间点，以异步方式生长的细胞的孵育用BrdU在S期标记细胞，标记的细胞追逐不同​​的时间（通常为4-6小时）。大多数标记的细胞将在脉冲后立即处于S期。追逐的长度被选择为使得追后，标记细胞将在晚S期，G2或有丝分裂，其可以通过形态特征中心体相对于NUC如 - 位置进行验证雷，中心体之间的距离，染色体等的缩合因此，追逐的长度取决于为S，G2和特定细胞类型的M期的平均持续时间。由于这种方法避免了细胞周期抑制剂，如羟基脲，阿非迪霉素，诺考达唑等 ，其允许更生理学相关细胞周期分析。 因此，我们在这里表明，单独的定量荧光显微术测定中，或在用BrdU脉冲追踪试验组合，是一种简单而功能强大的技术中的未扰动的细胞周期，以精确地测量在候选蛋白质的中心体水平的相对变化。我们测量VDAC3，我们在中心体最近确定除了线粒体16,28的蛋白质的中心体的水平，使用这些测定。在这里所得到的结果验证我们之前的看法，即VDAC3的中心体游泳池被降解调节，也各有不同的细胞周期dependen吨方式16中 ，还证实了该方法的适用性。

<table border="0" cellpadding="0" cellspacing="0" width="576">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Name&#160;</td>
			<td style="width:105px;">Company&#160;</td>
			<td style="width:99px;">Catalog number</td>
			<td style="width:195px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">Fibronectin</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">F8141</td>
			<td style="width:195px;">Stock of 1 mg/ml in water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">DMSO</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">D2650</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">MG115</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">SCP0005</td>
			<td style="width:195px;">Stock of 10 mM in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:179px;">BrdU</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">B5002</td>
			<td style="width:195px;">Stock of 10 mM in DMSO</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Anti-g-tubulin (mouse monoclonal, clone GTU 88)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T 6557</td>
			<td style="width:195px;">1:200 in IIF blocking buffer&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Anti-VDAC3 (rabbit polyclonal)&#160;</td>
			<td style="width:105px;">Aviva Systems Biology</td>
			<td style="width:99px;">ARP35180-P050</td>
			<td style="width:195px;">1:50 in IIF blocking buffer, 1:1000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-Sas6 (mouse monoclonal)&#160;</td>
			<td style="width:105px;">Santa cruz biotechnology</td>
			<td style="width:99px;">sc-81431</td>
			<td style="width:195px;">1:100 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-Cep135 (rabbit polyclonal)</td>
			<td style="width:105px;">Abcam</td>
			<td style="width:99px;">ab-75005</td>
			<td style="width:195px;">1:500 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-BrdU (rat monoclonal)&#160;</td>
			<td style="width:105px;">Abcam</td>
			<td style="width:99px;">ab6326</td>
			<td style="width:195px;">1:250 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Alexa Fluor 350 Goat Anti-Rat IgG (H+L)</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21093</td>
			<td style="width:195px;">1:200 in IIF blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21202</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21203</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:179px;">Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21206</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A21207</td>
			<td style="width:195px;">1:1000 in IIF blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-g-tubulin (rabbit polyclonal)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T5192</td>
			<td style="width:195px;">1:1000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Anti-a-tubulin (mouse monoclonal, DM1A)</td>
			<td style="width:105px;">Sigma</td>
			<td style="width:99px;">T9026</td>
			<td style="width:195px;">1:20000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">A10043</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Mouse IgG (H&#38;L) Antibody IRDye800CW Conjugated</td>
			<td style="width:105px;">Rockland antibodies</td>
			<td style="width:99px;">610-731-002</td>
			<td style="width:195px;">1:10000 in WB blocking buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">SlowFade Gold Antifade Reagent&#160;</td>
			<td style="width:105px;">Life technologies</td>
			<td style="width:99px;">S36936</td>
			<td style="width:195px;">Mounting media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Round coverslips 12CIR.-1</td>
			<td style="width:105px;">Fisherbrand</td>
			<td style="width:99px;">12-545-80</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Olympus IX-81 microscope</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Retiga ExiFAST 1394 IR camera</td>
			<td style="width:105px;">QImaging&#160;</td>
			<td style="width:99px;">32-0082B-238</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">100X Plan Apo oil immersion objective</td>
			<td style="width:105px;">Olympus</td>
			<td style="width:99px;"></td>
			<td style="width:195px;">1.4 numerical aperture</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:179px;">Slidebook software package</td>
			<td style="width:105px;">Intelligent Imaging Innovations</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Odyssey IR Imaging System</td>
			<td style="width:105px;">Li-cor Biosciences</td>
			<td style="width:99px;"></td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:179px;">Bicinchoninic acid (BCA) assay</td>
			<td style="width:105px;">Thermo Scientific</td>
			<td align="right">23227</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow UV cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M622</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Blue cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M643</td>
			<td>Filter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U-MNU2 Narrow Green cube</td>
			<td style="width:105px;">Olympus</td>
			<td>U-M663</td>
			<td>Filter</td>
		</tr>
	</tbody>
</table>

quantitative immunofluorescence assay to measure the variation in protein levels at centrosomes

中心体是充当有丝分裂纺锤体的两极保持基因组的完整性，或装配初级纤毛以促进感觉功能在细胞中的小但很重要的细胞器。一个蛋白的水平可在中心体比其他.cellular位置来调节不同的，并且在一些蛋白在不同点的细胞周期的中心体水平的变化似乎是中心粒装配的适当调节是至关重要的。我们开发了用于测量在一个蛋白的水平在中心体中固定的细胞来自不同样品的相对变化，在细胞周期的不同阶段，例如，或用各种试剂处理后的定量荧光显微术测定。这个测定的原理在于测量对应于蛋白质在一个小区域的背景校正的荧光强度，和正常化了的测量对相同的对不选择的实验中c而变化的另一蛋白质ondition。利用该测定在用BrdU脉冲和追策略学习未扰动细胞周期的组合，我们已定量具体地说在中心体验证我们最近的观察结果VDAC3的中心体池在细胞周期中被调节在中心体，可能是由蛋白酶体介导的降解。

我们最近的研究中发现VDAC3，线粒体孔蛋白16,28中的一个新的中心体定位和功能。几种哺乳动物细胞，包括使用VDAC3特异性抗体RPE1细胞的免疫染色表明突出中心体染色和较弱线粒体染色。我们还发现，中心体VDAC3优先与母亲中心粒，而无论是内源性的中心体池关联和异位表达VDAC3被降解16规范。为了验证定量的IIF分析我们检查了在S期的变化，中心体相关VDAC3池中的细胞。 <p class="jo...

Watch this Scientific Journal Video about Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes at JoVE.com

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-immunofluorescence-assay-to-measure-variation-protein

Research

JoVE Journal

Biology

14.9K 次观看.  The Ohio State University. Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard. 

视频: Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

科研

生物学

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

漂白实验（FRAP和FLIP）来衡量生活胚胎干细胞的染色质的蛋白质动力学

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

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 ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

在飞睾丸成像中心体

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...