DF是由美国国家科学基金会桥梁博士奖学金的支持。在这项工作中所使用的显微镜​​是由科罗拉多州立大学的显微镜成像网络核心基础设施授予部分支持。在作者的实验室的网格蛋白适配器的工作是支持CSU的启动资金和美国心脏协会奖09SDG2280525到SD

1。 SLA1基因选择标记的GFP标记和团以融合的GFP标记直接SLA1基因的开放阅读框3&#39;（Sla1p彗星端），同时标记基因与大肠杆菌应用Longtine方法17 大肠杆菌根R基因，允许G418筛选。 <ol><li>生成一个用PCR的DNA片段作为模板使用的pFA6a - GFP的质粒（S65T）kanMX6 18及以下的引物：正向，5&#39; - CA AGG民航局海湾合作委员会的AAC阿拉木图TTC亚洲空运中心GCT公司的ACT GCA TCA的学能测验CCG TTT GGA TTC CGG ATC CCC GGG TTA ATT的AA - 3&#39;和反向，5&#39; - CA达AGC TTG TTT豪TTA TTA台泥达AAA级ATC TTA AAA级交咨会ATT亚洲空运中心 GAA TTC聚糖反恐委员会GTT TAA的AC - 3“。下划线序列对应的SLA1紧接（正向引物）和终止密码子（反向引物）的基因的特定部分。隔离的DNA纯化的PCR产物经琼脂糖凝胶电泳。 </li><li>准备50毫升文化南 。在早期对数生长期（OD 600 = 0.2-0.6） 的酵母SEY6210应变（MAΤαURA3 - 52，LEU2 - 3，112 HIS3Δ200，TRP1Δ901，lys2 - 801，suc2Δ9的GAL - MEL）19日 ，在YPD增长。在室温下的附带在2000 XG 3分钟，用无菌水洗两次。 </li><li>变换使用醋酸锂程序20细胞在步骤1中获得的PCR片段。用1 ml无菌水清洗细胞，加入1毫升YPD和孵育4小时，30 °，一个摇床中的C。 </li><li>传播YPD - G418的板细胞选择G418抗性的转化，在30 ° C孵育2-3天。 YPD - G418的板挑上的殖民地和他们连胜。 </li><li>使用殖民地- PCR检测，确定其中GFP根模块集成与SLA1基因序列同源重组转化。使用正向引物，内GFP根模块内的SLA1开放阅读框和反向引物退火的退火。确定殖民地PCR产物预期大小，并通过测序验证。 </li><li>屏幕荧光显微镜下的殖民地，以确认他们有绿色荧光。 </li></ol> 2。在SLA1基因突变变异网格蛋白盒（VCB） <ol><li>介绍AAALQ在以下两个步骤的方法15 SLA1基因（2407至2415年个核苷酸）突变LLDLQ。 </li><li>首先，健全SLA1核苷酸的PCR扩增1407至10年和2427年至2589年，克隆的片段，分别到pBluescriptKS通知/酶BamHI和EcoRI /贝网站。 </li><li>亚克隆到的酶BamHI / EcoRI位点的PCR片段含有URA3。 </li><li>切割与NotI /贝重组质粒，隔离凝胶净化URA3片段，并引进锂醋酸转化成Sla1 - GFP株产生的第1部分中，或到TVY614（MATA URA3 - 52 LEU2 - 3112 HIS3Δ200TRP1 Δ901- 801 lys2 suc2 -Δ9pep4：LEU2 prb1：HISG PRC1：HIS3）21。 </li><li>传播辅以缺乏尿嘧啶的SD板的细胞。确认菌落PCR和测序，市建局+殖民地包含适当的综合URA3取代VCB片段。 </li><li>在第二个步骤，亚克隆SLA1核苷酸1207年至2589年，到pBluescriptKS NotI /贝网站。使用快速转换- XL站点定向诱变试剂盒（Stratagene公司），变异的VCB残留LLDLQ AAALQ。通过测序验证。 </li><li>切割与BSGI /阿给的建设，并隔离含有凝胶净化片段突变VCB。 Cotransform到2.3部分所获得的应变pRS313（HIS3）22 BSGI /阿给的片段。 </li><li>传播为辅的SD板缺乏组氨酸的细胞。他副本板+克隆到琼脂含5 fluorotic酸，以确定细胞突变的序列中，取代URA3，从而再生sla1基因包含LLDLQ AAALQ突变（sla1 AAA级 ）的媒介。菌落PCR和测序确认。 </li></ol> 3。荧光显微镜<ol><li>成长的酵母菌株，直至到达一个外径600 = 0.1-0.4 Sla1 - GFP和sla1 AAA - GFP的4毫升补充SD媒体1和2在30 ° C在黑暗中转子中产生。旋转离心于4000在室温下1分钟XG文化1毫升。丢弃〜950μL上清。在剩余的液体（〜50μL），重悬细胞。 </li><li>存款3μL的细胞悬液到显微镜幻灯片，并与玻璃盖玻片覆盖。轻保护样品。 </li><li>登上一个100X油浸目标的一个旋转盘共聚焦显微镜幻灯片。 </li><li>找到正确的使用适当的过滤器明照明或488 nm激光激发和检测从GFP荧光的细胞焦平面。 </li><li>捕捉时间推移图像Sla1 - GFP和sla1 AAA - GFP的细胞在500毫秒的曝光时间（或适当的值）ñ的时间间隔为150秒每秒一个形象。预期结果：绿色荧光蛋白标记punctae限制细胞膜的内表面上会出现，持续几秒钟，向细胞中心的移动信号很快消失（表明外套拆卸）。 </li><li>选择部分在这点看到的形象出现，停留几秒钟，并消失。剪裁的图像，每帧的时间推移图像。 </li><li>表示此节在每150秒的时间推移图像沿轴对应的时间过去了，通过调整帧帧的图像生成kymograph。 </li><li>计算基于多少秒（时间推移帧）的斑点在kymograph每个点的时间。注意每个点应该向细胞内部的“曲线”正在消失，反映了内吞作用和拆卸的外衣。 </li><li>比较sla1 AAA - GFP的野生型Sla1 - GFP。重复结果，以确定是否有统计学意义。预期结果：sla1 AAA - GFP的点仍然存在明显长于野生型，表明在大衣形成 15的缺陷（〜30秒）（〜80秒）。 </li></ol> 4。酵母细胞质提取物和总细胞提取物的制备<ol><li>接种用适当的酵母菌株，如TVY614 21 3公升的YPD，增长30 ° C，直到到达一个外径 600 = 1-2，在摇床孵化器。 </li><li>酵母培养物在室温下离心20分钟3,700 X克。弃上清，加无菌水3-5毫升，吸管向上和向下，直到沉淀完全重悬。 </li><li>倒入液氮150毫升250毫升塑料烧杯中。闪存冻结逐滴加入到液态氮中的圆形图案，以避免冷冻颗粒凝集酵母。不要让颗粒解冻，如有必要，添加更多的液氮。 </li><li>浇在液氮冷藏一个不锈钢搅拌器容器。允许液氮几乎完全蒸发。添加冷冻酵母颗粒冷搅拌器容器。冷橡皮塞关闭搅拌器容器和酵母颗粒研磨10秒。反转容器的3-4倍混合内容和重复两次研磨的步骤。地面冻结酵母将有粉末状的外观。 </li><li>用液氮冷藏一个漏斗和一个50毫升的锥形管。使用冷漏斗，地面酵母转移到管。冻土酵母可存放于-80 ° C或立即使用。 </li><li>为了获得GST融合蛋白的亲和力检测（第五部分），重3克冻土TVY614酵母在15毫升的锥形管，并在室温缓冲区3毫升悬浮的胞质提取一（10毫米HEPES，pH值7.0， 150 mM氯化钠，1 mM的EDTA，1毫米的数码地面电视）含有蛋白酶抑制剂的鸡尾酒（Sigma公司）。 </li><li>第反转管几次，直到完全解冻，然后在冰上。 </li><li>超速离心机的提取物在4 ° C为20分钟30万小工小心转移上清液（细胞质提取物），一个锥形管，用吸管，而不会干扰颗粒。保持在冰上的细胞质中提取。 </li><li>加入100μL50％（V / V），谷胱甘肽琼脂糖凝胶缓冲液A中的水泥浆，很方便削减提示结束，以便pipeting珠。旋转4 ° C下15分钟，2分钟，1000 XG，转移上清液（细胞质提取）到一个新的管降速4离心珠° C。储备50μL提取输入控制。 </li><li>要获得免疫共沉淀实验的总细胞提取物（6），使用TVY614株（WT SLA1），AAALQ TVY614背景中的第2部分中产生的突变携带 LLDLQ sla1 AAA级应变，和一个背着删除sla1应变SLA1基因，如GPY3130 23。相应的锥形管冻土酵母和重量2克重悬在2毫升室温缓冲区，含有蛋白酶抑制剂的鸡尾酒（Sigma）和2％的Triton X - 100的。 </li><li>第和反转管几次，直到完全解冻，然后在冰上孵育10分钟，定期反演混合。 </li><li>传输材料的离心管和旋转4 ° C下15分钟在16000 XG（微量的最高速度）。小心将上清转移（总细胞提取物）使用冰吸管一个锥形管，而不会干扰颗粒。 </li><li>添加100μL50％（V / V）的蛋白质在缓冲液A -琼脂糖凝胶浆旋转4 ° C下15分钟，降速离心珠在4 ° C为2分钟在1000 XG和转让上清液（总提取物）到一个新的管。储备50μL提取输入控制。 </li></ol> 5。 GST融合蛋白的亲和力检测<ol><li>健全由PCR扩增一个片段containi吴Sla1p变种网格蛋白盒（VCB）（残基803-807），克隆到PGEX - 5X获得PGEX - 5X - VCB。通过测序验证。 </li><li>使用PGEX - 5X - VCB作为模板的快速转换- XL网站，定向诱变试剂盒（Stratagene公司），变异VCB残留LLDLQ AAALQ获得PGEX - 5X - vCBmut。通过测序验证。 </li><li>快速消费税和融合蛋白GST - VCB，并在大肠杆菌中的GST - vCBmut 大肠杆菌 （BL21 DE3），净化珠谷胱甘肽谷胱甘肽琼脂糖凝胶，洗脱，透析对PBS，并确定蛋白浓度。 </li><li>标签3离心管：商品及服务税，消费税，VCB，GST - vCBmut，并加入1 ml的PBS缓冲液中谷胱甘肽琼脂糖浆50％（V / V）30μL，和50微克的dialized商品及服务税，消费税VCB或GST - vCBmut融合蛋白。 </li><li>在室温为30分钟，旋转，让有约束力的。 </li><li>珠洗净，用PBS缓冲液A的2倍和1次加入1毫升酵母细胞质提取物 - 准备在第4部分所述 - 每管。 </li><li>旋转管，在5000 XG颗粒的珠子1小时在4 ° C，旋转10秒，弃上清，并迅速用缓冲液洗珠子的3倍缓冲液A含0.1％Triton X - 100的的，1时间</li><li>离心更多的时间和使用的凝胶加载提示消除尽可能多的液体珠。此时样品可以储存在-20 ° C，继续在以后的时间。 </li><li> 2X Laemmli样品缓冲液加入15μL每管孵化，在96℃5分钟，并在5000旋转10秒小工</li><li>使用网格蛋白重链抗体免疫印迹分析的样品。预期结果：网格蛋白GST - VCB结合，但不GST或GST - vCBmut。还通过SDS - PAGE分析的样本，以确认GST融合蛋白的类似加载。 </li></ol> 6。共免疫实验<ol><li>标签3离心管：WT（野生型SLA1），sla1 AAA级 ，并sla1Δ，并添加1毫升的PBS，30μL，和2微克的50％（V / V）缓冲液中蛋白质A -琼脂糖凝胶浆兔抗Sla1p抗体。 </li><li>在室温为30分钟旋转。 </li><li>洗磁珠用PBS和1次用缓冲液含1％TRITON X - 100的两倍。 </li><li>加入1毫升相应的总酵母提取准备在第4部分：SLA1，sla1 AAA级 ，并sla1Δ。旋转1小时在4 ° C </li><li>旋转10秒在5000 XG颗粒珠，弃上清液，并迅速用缓冲液洗珠子的3倍，含0.1％TRITON X - 100，并用缓冲液A 1次</li><li>离心更多的时间和使用的凝胶加载提示消除尽可能多的液体珠。此时样品可以储存在-20 ° C，继续在以后的时间。 </li><li> 2X Laemmli样品缓冲液加入15μL每管孵化，在96℃5分钟，并在5000旋转10秒小工</li><li>使用网格蛋白重链抗体免疫印迹分析的样品。预期结果：网格蛋白联合免疫与野生型Sla1p sla1 AAA，或在sla1Δ样品，但没有到。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2352/2352fig1.jpg" alt="图1" /> 图1。Sla1p网格蛋白适配器吞网站由活细胞荧光显微镜分析。一帧（左）从视频和相应的kymographs表达Sla1 - GFP或sla1 AAA级 GFP携带LLDLQ AAALQ突变的酵母细胞（右） 。野生型和突变Sla1 - GFP的表达从内源性SLA1轨迹。吞站点观察到分布在细胞周边（左图像）的亮点。白线之间的区域对应的kymographs从产生区域。电影在1帧/秒的帧速率。请注意在AAALQ突变（sla1 AAA级）LLDLQ Sla1 - GFP相比，野生型（SLA1）的寿命更长。 <img src="/files/ftp_upload/2352/2352fig2.jpg" alt="图2" /> 图2网格蛋白和Sla1p变网格蛋白框之间的物理相互作用。 GST融合Sla1p势必谷胱甘肽- Sepharose树脂和培养一个从野生的细胞质中提取片段aa798 - 813包含序列LLDLQ（GST - VCB），相应的AAALQ突变（GST - vCBmut），或单独商品及服务税（消费税）型酵母细胞。洗脱和分析相关蛋白免疫印迹（IB），网格蛋​​白重链。

<ol>
	<li>Mellman, I., Warren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+road+taken:+past+and+future+foundations+of+membrane+traffic.">The road taken: past and future foundations of membrane traffic.</a> Cell. 100, 99-112 (2000).</li><li>Engqvist-Goldstein, A. E., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+assembly+and+endocytosis:+from+yeast+to+mammals.">Actin assembly and endocytosis: from yeast to mammals.</a> Annu Rev Cell Dev Biol. 19, 287-332 (2003).</li><li>Conner, S. D., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+portals+of+entry+into+the+cell.">Regulated portals of entry into the cell.</a> Nature. 422, 37-44 (2003).</li><li>Bonifacino, J. S., Traub, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signals+for+sorting+of+transmembrane+proteins+to+endosomes+and+lysosomes.">Signals for sorting of transmembrane proteins to endosomes and lysosomes.</a> Annu Rev Biochem. 72, 395-447 (2003).</li><li>Ungewickell, E. J., Hinrichsen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis:+clathrin-mediated+membrane+budding.">Endocytosis: clathrin-mediated membrane budding.</a> Curr Opin Cell Biol. 19, 417-425 (2007).</li><li>Doherty, G. J., McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+endocytosis.">Mechanisms of endocytosis.</a> Annu Rev Biochem. 78, 857-902 (2009).</li><li>Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin.">Clathrin.</a> Annu Rev Biochem. 69, 699-727 (2000).</li><li>Brodsky, F. M., Chen, C. Y., Knuehl, C., Towler, M. C., Wakeham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+basket+weaving:+formation+and+function+of+clathrin-coated+vesicles.">Biological basket weaving: formation and function of clathrin-coated vesicles.</a> Annu Rev Cell Dev Biol. 17, 517-568 (2001).</li><li>Owen, D. J., Collins, B. M., Evans, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptors+for+clathrin+coats:+structure+and+function.">Adaptors for clathrin coats: structure and function.</a> Annu Rev Cell Dev Biol. 20, 153-191 (2004).</li><li>Dell'Angelica, E. C., Klumperman, J., Stoorvogel, W., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+AP-3+adaptor+complex+with+clathrin.">Association of the AP-3 adaptor complex with clathrin.</a> Science. 280, 431-434 (1998).</li><li>Dell'Angelica, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-binding+proteins:+got+a+motif?+Join+the+network!.">Clathrin-binding proteins: got a motif? Join the network!.</a> Trends Cell Biol. 11, 315-318 (2001).</li><li>Holtzman, D. A., Yang, S., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic-lethal+interactions+identify+two+novel+genes,+SLA1+and+SLA2,+that+control+membrane+cytoskeleton+assembly+in+Saccharomyces+cerevisiae.">Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae.</a> J Cell Biol. 122, 635-644 (1993).</li><li>Howard, J. P., Hutton, J. L., Olson, J. M., Payne, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sla1p+serves+as+the+targeting+signal+recognition+factor+for+NPFX(1,2)D-mediated+endocytosis.">Sla1p serves as the targeting signal recognition factor for NPFX(1,2)D-mediated endocytosis.</a> J. Cell. Biol. 157, 315-326 (2002).</li><li>Mahadev, R. K., Pietro, S. M. D. i., Olson, J. M., Piao, H. L., Payne, G. S., Overduin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Sla1p+homology+domain+1+and+interaction+with+the+NPFxD+endocytic+internalization+motif.">Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif.</a> EMBO J. 26, 1963-1971 (2007).</li><li>Pietro, S. M. D. i., Cascio, D., Feliciano, D., Bowie, J. U., Payne, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+clathrin+adaptor+function+in+endocytosis:+A+novel+role+for+the+SAM+domain.">Regulation of clathrin adaptor function in endocytosis: A novel role for the SAM domain.</a> EMBO J. 29, 1033-1044 (2010).</li><li>Kaksonen, M., Toret, C. P., Drubin, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+design+for+the+clathrin-+and+actin-mediated+endocytosis+machinery.">A modular design for the clathrin- and actin-mediated endocytosis machinery.</a> Cell. 123, 305-320 (2005).</li><li>Longtine, M. S., McKenzie, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+modules+for+versatile+and+economical+PCR-based+gene+deletion+and+modification+in+Saccharomyces+cerevisiae.">Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.</a> Yeast. 14, 953-9561 (1998).</li><li>Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C., Philippsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+HIS3+marker+and+GFP+reporter+modules+for+PCR-targeting+in+Saccharomyces+cerevisiae.">Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae.</a> Yeast. 13, 1065-1075 (1997).</li><li>Robinson, J. S., Klionsky, D. J., Banta, L. M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+sorting+in+Saccharomyces+cerevisiae:+isolation+of+mutants+defective+in+the+delivery+and+processing+of+multiple+vacuolar+hydrolases.">Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.</a> Mol Cell Biol. 8, 4936-4948 (1988).</li><li>Ito, H., Fukuda, Y., Murata, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+intact+yeast+cells+treated+with+alkali+cations.">Transformation of intact yeast cells treated with alkali cations.</a> J. Bacteriology. 153, 163-168 (1983).</li><li>Vida, T. A., Emr, S. 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+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Sikorski, R. S., Hieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+of+shuttle+vectors+and+yeast+host+strains+designed+for+efficient+manipulation+of+DNA+in+Saccharomyces+cerevisiae.">A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.</a> Genetics. 122, 19-27 (1989).</li><li>Piao, H. L., Machado, I. M., Payne, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NPFXD-mediated+endocytosis+is+required+for+polarity+and+function+of+a+yeast+cell+wall+stress+sensor.">NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor.</a> Mol Biol Cell. 18, 57-65 (2007).</li></ol>

网格蛋白包被小泡（CCV）参与内吞作用和跨高尔基网络运输和内涵体，保守的途径是真核细胞生物学的基础。此方法文件中所描述的方法是有用的负责CCV的形成，特别是适配器蛋白和网格蛋白之间的相互作用的分子机制研究。 GST融合蛋白的亲和力和免疫共沉淀实验，让测试适配器（候选人）和网格蛋白之间的物理协会。绿色荧光蛋白标记和荧光显微镜成像允许在活细胞的动态研究。尤其是...

<table border="1"><th>材料</th><th>公司</th><tbody><tr><td>快速转换- XL定点突变试剂盒</td><td> Stratagene公司</td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td>西格玛</td></tr></tbody></table>

in vivo and in vitro studies of adaptor-clathrin interaction

一个主要的内吞途径启动，形成网格蛋白包被小泡（CCVS），从细胞表面到内涵体运输货物1-6。 CCVS的区别在于一个多面体的笼形蛋白，大衣囊膜的晶格，并担任机械支架。从个别网格蛋白triskelia，三个重型和轻链亚基7,8组成的笼形蛋白的可溶形式在囊泡形成网格蛋白大衣组装。由于triskelion没有直接绑定到膜的能力，网格蛋白结合适配器协会通过与血脂和/或膜蛋白9的链接，形成网格蛋白晶格膜的关键。适配器还包跨膜蛋白的货物，如受体，而且可以互相交流，与其他组件的CCV形成机械 9 ​​。 有超过20个网格蛋白适配器，一些参与网格蛋白介导的内吞作用和其他本地化跨高尔基网络或内涵体 9 。 HIP1R（酵母Sla2p）的异常，所有已知的网格蛋白适配器绑定到网格蛋白重链 9 N -末端螺旋桨域。网格蛋白适配器非结构化灵活的连接器连接的折叠域组成的模块化的蛋白质。在这些连接器的地区中，短期结合基序调解的网格蛋白的N端结构域或其他组件的囊泡形成机械9 ​​的交互。两个不同的网格蛋白结合的图案已被定义：网格蛋白框和框的W - 9 。的共识网格蛋白- box序列的最初定义为L [L / I] [D / E / L / F]，[D / E] 10，但变种已被后来发现11。符合的W - box序列PWxxW（其中x是任何残留物）。 Sla1p（与肌动蛋白结合蛋白1合成致命的）最初确定为一个肌动蛋白相关蛋白在酵母细胞的12内吞网站是正常的肌动蛋白细胞骨架的结构和动力学的必要。 Sla1p还结合NPFxD吞分拣信号NPFxD信号13,14轴承货物的内吞作用的关键。最近，Sla1p被证明绑定通过类似网格蛋白中，LLDLQ的一个Motif网格蛋白，被称为网格蛋白盒（VCB）的一个变种，和功能吞网格蛋白适配器15。此外，Sla1p已成为广泛使用的标记为在活细胞荧光显微镜研究 16的内吞大衣。这里我们使用Sla1p作为一个模型来描述适配器网格蛋白的相互作用研究方法。我们重点对活细胞的荧光显微镜，消费税拔下来，和免疫共沉淀方法。

Watch this Scientific Journal Video about In vivo and in vitro Studies of Adaptor-clathrin Interaction at JoVE.com

In vivo and in vitro Studies of Adaptor-clathrin Interaction

网格蛋白介导的内吞作用取决于适配器的蛋白质，协调货物选择和网格蛋白外衣大会。在这里，我们描述的程序来研究网格蛋白适配器的物理互动和活细胞成像的方法，作为一种模式使用酵母吞适配器蛋白Sla1p。

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

in-vivo-and-in-vitro-studies-of-adaptor-clathrin-interaction

Research

JoVE Journal

Biology

In vivo and in vitro Studies of Adaptor-clathrin Interaction

14.4K 次观看.  Colorado State University. 网格蛋白介导的内吞作用取决于适配器的蛋白质，协调货物选择和网格蛋白外衣大会。在这里，我们描述的程序来研究网格蛋白适配器的物理互动和活细胞成像的方法，作为一种模式使用酵母吞适配器蛋白Sla1p。

视频: In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

科研

生物学

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

细胞质DNA的检测途径的刺激<em&gt;在体外</em&gt;和<em&gt;体内</em

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

检测植物-真菌相互作用中果胶的双重染色方法

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

使用体外和体内模型分析细胞外囊泡介导的血管钙化

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

通过脱细胞定量骨骼肌脂肪浸润

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

作者聚焦：基于脱细胞化的骨骼肌脂肪浸润定量  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.