我们感谢克里·布卢姆（北卡罗莱纳大学），凯·李（NCI），史蒂文·马库斯（科罗拉多州立大学）和埃尔马·希贝尔（海德堡大学）分享各种FP-TUB1质粒。我们是在滑动室制备方法训练我们感谢梅利莎·加德纳（明尼苏达大学）。这项工作是由美国国立卫生研究院（NIH）授予R01GM112893-01A1（以JKM）和T32GM008730（到CE）的支持。

1.准备-Leu降板<ol><li>添加800毫升的无菌去离子水（DIH 2 O）到2L烧瓶中。添加26.71克漏失碱（DOB）粉末，0.69克CSM-的Leu，和20g琼脂。用磁力搅拌棒混合。带来至1L DIH 2 O. </li><li>高压釜在121℃和19磅30分钟。 </li><li>除去从高压釜的烧瓶中，并使其冷却至〜65℃，温和搅拌。 </li><li>倾30毫升/板放入100mm直径板。让这些坐在室温下36-48小时，在4℃下储存之前。 </li></ol> 2.整合GFP-TUB1的GFP标记的微管蛋白的组成型表达<ol><li>煮沸的10毫克/毫升的单链DNA（ssDNA）的储备浓度3分钟。 </li><li>结合煮沸ssDNA的2μL与400ng的纯化GFP-TUB1质粒DNA 11。 注:有多种质粒可用于稳定，荧光标记在酵母中利用替代的荧光团和选择性标记微管。其中一些替代品的讨论部分描述。 </li><li>的DNA混合物中加入感受态酵母细胞的50微升等分试样。 注:准备感受态酵母细胞与山梨糖醇溶液（SORB; 100mM的LiOAc，10mM的Tris-盐酸pH为8，1mM EDTA中的pH 8，和1M山梨糖醇，用稀乙酸调节至pH 8）。制备感受态酵母细胞的详细描述，请参见参考12。 </li><li>涡彻底。 </li><li>加入6倍的聚乙二醇（PEG）溶液（100mM的LiOAc，10mM的Tris-盐酸pH为8，1mM EDTA中/ NaOH调节pH为8，和40％PEG3350）至DNA-细胞混合物的总体积。 </li><li>涡彻底。 </li><li>孵育在30℃下温和搅拌至少1小时。 </li><li>二甲基亚砜（DMSO）添加到总体积和涡流的10％。 </li><li>孵育在42℃下15分钟。 </li><li>离心机中以2500×g离心5分钟。 </li><lI&gt;去除上清，悬浮细胞在100μLDIH 2 O的</li><li>板单元上的压降-Leu板。 注:GFP-TUB1结构中含有亮氨酸营养缺陷型标记，而其他的构造可以使用一个替代指标。漏失介质应该是特定于构建体的营养缺陷型标记。 </li><li>允许转化的细胞在30℃下3-5天生长。 </li><li>在新鲜-Leu漏失板重新条纹单一转化菌落通过使用高压灭菌牙签转移菌落。过夜孵育所述板在30℃下。 </li><li> 通过从单菌落从平板在步骤2.14中的水2μL，在一个干净的载玻片悬浮大约1细胞μL目视筛选每个转化为GFP信号。覆盖悬浮细胞用盖玻片并用荧光显微镜观察。 <ol><li>激发转化用488nm的光，并寻找一个GFP信号的存在。 <br/&gt;注:此步骤可以使用一个旋转盘共聚焦显微镜，但配备有100X物镜和用于成像GFP就足够了适当光学一个宽视场显微镜。没有可见的GFP标记的微管，或那些具有异常高的荧光细胞，应被拒绝。在此之后，酵母菌株已准备好为用于成像培养。 </li></ol></li></ol> 3.成长液体酵母培养物<ol><li>接种的酵母细胞的〜1μL从单菌落上在3mL的合成完全培养基的板。使细胞以250rpm振荡在30℃下生长过夜。 </li><li>稀释饱和培养翌日所述介质中的&lt;0.1 OD 600。返回稀释的培养到30℃的振荡3-4小时或直至OD 600为0.4和0.8之间，当细胞准备被装载到成像室中。 </li></ol> 4.准备流室幻灯片<ol><li>切在长石蜡膜在宽的片材成片4和7。 </li><li>放置一个标准显微镜载玻片上纵向石蜡膜的4英寸的宽度和缠绕3-4次，使得滑动被覆盖在膜是3-4层厚的载玻片上的膜。按层一起，以确保有一个合理的密封;这将使它更容易削减。请勿按压太硬或使膜起皱。 </li><li>切割沿使用清洁盒切刀剃刀滑动件的外侧边缘上的石蜡膜。使用另一张幻灯片，以用于切割提供直边。 </li><li>剥离多余的膜从上述工作滑动并丢弃。的膜的剩余堆叠将覆盖滑动的一个面，将被用来制造成像腔室之间的壁。 </li><li>使用所述第二幻灯片作为直边，切割堆叠的薄膜层沿着滑动的长轴成大约10条，每2-4毫米宽。 </li><li>垂直地切断层叠石蜡薄膜层到在步骤4.5制成，沿着滑动的短轴的切口，以创建条2-4毫米宽，23-24毫米长，和3-4层厚。 </li><li>剥离使用剃刀以45°角切割的条带。使用镊子，均匀地分布在一个干净的显微镜载片切割薄膜条创建的腔室所需的数字。至多3个腔室（带4个石蜡膜带制成）可在一个载片被创建。 </li><li>放置的薄膜条带的顶部上的单盖玻片并将其与镊子移动到位。 </li><li>放置准备好的滑动，盖玻片起来，在设定在95〜℃的加热板上（这大约是在大多数电炉低介质设置）。加热滑动直到薄膜条带是半透明的（大约3分钟），然后密封所述滑动件和由与所述镊子盖玻片轻轻按压盖玻片一起。 注意:这是仅在直接上方的石蜡膜条带盖玻片的区域按重要的，因为刮擦上述c的区域hambers会降低图像质量。小心不要过热幻灯片;汽化的薄膜将涂覆室的内部有防止细胞粘附的疏水性膜。 </li><li>使用镊子从加热板除去所述滑动件（滑动会热）并放在一边与盖玻片面朝上冷却;作为滑动冷却时，该膜将返回到白色，不透明的着色。 注意:此时，幻灯片已经准备好装载培养的酵母细胞。额外的幻灯片，可向这一点，并存储在一个干净，干燥的地方无限期量。 </li></ol> 5.安装酵母细胞制备成流室幻灯片<ol><li>解冻伴刀豆球蛋白A（2毫克/毫升在无菌DIH 2 O）的冻结，将200μL等分试样。 </li><li>通过吸取到开放端中的一个添加约100μL解冻伴刀豆球蛋白A的所制备的载玻片的每个室。加入足够的伴刀豆球蛋白A，以填补室。对于这个STEP，在伴刀豆球蛋白A将通过经由毛细管作用室中拉出。 </li><li>挂滑动，盖玻片向下两个离心管架或笔之间5分钟，以允许所述伴刀豆球蛋白A粘附在盖玻片（ 即，腔室）。 </li><li> 返回幻灯片盖玻片上位置。用2x洗涤腔室中的腔室体积（在步骤5.2，以上确定的;约200微升）的合成完全培养基的除去伴刀豆球蛋白A. <ol><li>通过腔室的流动介质，使用纸巾的任角落或对半折叠滤纸以将流体从室的一端的同时吸取新鲜流体到另一个。可替代地，使用真空吸气器仔细吸取流体从一端的同时吸取新鲜流体到另一个。 </li></ol></li><li>称重传感器通过使用定向流流动方法具2倍的细胞悬浮液（来自步骤3.4）的腔室容积（约200μL）D IN步骤5.4.1说明。 注意:我们的目标是在图像盖玻片细胞的单层。因此，加载细胞足够小，它们不会桩上彼此的顶部，但足够大，足够的细胞存在于字段来收集每图像采集的最大数据量的浓度是重要的。细胞的腔室中的最佳浓度可以变化，并应根据经验确定。为了提高细胞的浓度在流动室，在1,000×g下轻轻沉淀细胞培养物2分钟，并除去一些上清液。为了降低细胞的浓度，新鲜的合成完全培养基添加到细胞悬浮液。细胞的浓度可通过在相差显微镜检查室（见下文）步骤5.7之后进行评估。在这一点上，附加的细胞可以通过在多种细胞悬浮液流向被添加到腔室中。然而，减少细胞密度在这一点上是不可行的，并最好地实现通过加载新的腔室用稀释的细胞悬浮液。 </li><li>挂在滑动盖玻片向下，如在步骤5.3所描述的，10分钟，以使细胞附着在盖玻片。 </li><li>由流过4倍的合成完全培养基的腔室容积（约400微升）冲洗掉未粘附的任何细胞。这将消除在腔室自由浮动的细胞，这可能污染图像采集。 </li><li>仔细干燥用纸巾的干净的角腔室的每个端部，使所述弯月面到盖玻片的边缘。 </li><li>密封用温水（75℃）密封剂室的每个端部（ 例如，VALAP;等份凡士林，羊毛蜡和石蜡）。 注意:此时，幻灯片准备图像。所述腔室可被成像多达9个小时，这取决于在每个室中的细胞密度。细胞将产生二氧化碳（CO 2）随着时间的推移，这将逐渐创建置换细胞气泡。 </li></ol><ol><li>使滑动到装有1.45 NA 100X CFI计划的Apo目的，压电阶段，旋转盘共聚焦扫描仪单元，照明激光器和照相机EMCCD倒置显微镜。维持阶段的在室温（25℃）采集期间的温度。 注意:高数值孔径允许更大的图像分辨率，并且压电阶段可实现高速Z堆叠采集。最小显微镜要求是100X物镜，照射激光，并且EMCCD相机。 </li><li>使细胞成为关注的焦点。确保采集字段具有通过搜索分组在一起细胞滑动聚类在一起的至少5-6细胞。 注意:虽然没有必要对图像多于一个小区更多的时间，成像多个小区在同一领域提高了数据采集的效率而不会损害数据质量。然而，这是关键细胞在同一焦平面，而不是堆积在彼此的顶部。 </li><li> 程序的多维采集随着时间的推移，收集多个z系列。 <ol><li>调节活性采集设置为以下范围内的设置:总Z深度= 6微米，以包含整个酵母细胞; Z-步长大小= 300纳米，以最大化光收集没有过采样;总持续时间= 10分钟;时间间隔= Z系列每4秒;和曝光时间=对于每个z平面90毫秒。 注:最佳曝光时间将根据照相机，激发光源，和其它因素而有所不同。通常，最佳曝光时间将是实现了3所需的最小时间:从GFP-标记的星体微管信号的1比从基于由所述测量EMCCD像素值细胞质到信号。 </li></ol></li><li>通过点击"运行"开始采集。允许它为完整的10分钟时间运行。保存数据作为图像系列（.TIFF或.nd2）。 </li><li>在室内选择一个新的领域，以图像，并重复步骤6.2-6.4。 注意:单个腔室应产生细胞的15和20之间的领域，根据腔室中的细胞密度。更高的细胞密度将产生较低的总的字段，作为CO 2在腔室将更快建立。 </li></ol> 7.图像分析<ol><li> 打开的ImageJ（图像文件<a href="https://imagej.nih.gov" target="_blank">https://imagej.nih.gov</a>通过使用生物格式进口商插件），为hyperstack。 <ol><li>开放的ImageJ并拖动从部分6所获取的图像堆栈直接到控制面板。生物格式进口商将自动启动。选择"OK"来加载图像堆栈作为hyperstack。 注意:"生物格式导入选项"的提示将打开，并确保"堆栈查看"部分，在"查看栈:"设置为"Hyperstack";和"堆叠顺序:"设置为"XYCZT。" </li></ol></li><li> 折叠每个Z系列到使用Z-项目功能的最大强度，二维投影。 <ol><li>选择"图像"菜单，在"堆栈"子菜单，"Z-项目"功能。从下拉列表中选择"最大投影"，然后单击"确定"。 </li></ol></li><li>应用中值滤波器的图像堆栈通过选择"过程"菜单，"过滤器"子菜单中，和"中值"功能来减少散斑噪声。输入2.0像素成方圆框，然后点击"确定"。 </li><li>确定在该领域前后期的细胞。 注意:这些的特征在于中型芽和短有丝分裂纺锤体，1-1.5微米的长度（ 图1;箭头指向一个短有丝分裂纺锤体）。在此阶段细胞是理想的用于分析的微管动力学，因为它们通常表现出仅一个FE发出的纺锤极7瓦特星体微管。星体微管能够被识别为表现出均匀的GFP信号强度从负端，在SPB，到正端，在细胞质中线性长丝。 </li><li>开始在图像序列的第一时间点，使用所分割的线路工具画沿着单个星体微管的管线，从负端，位于所述星体微管和更强烈标记纺锤体微管之间的连接处，向正端，这是在细胞质中（参见图1中的红线）。对微管的末尾双击即可完成分割线。 </li><li>记录使用"测量"功能通过按下键盘上的"M"按钮，在结果表中的测量。 注:其他的功能，如灰色的强度，可以通过在"分析"菜单中设置的测量和"设置测量"子菜单被记录。 </li><li>通过单击在Z滑块向右箭头或按下键盘上的"./&gt;"键前进到下一个时间点。重复步骤图像序列中的所有时间点的7.5和7.6。 </li><li>数据结果表中复制并粘贴到电子表格中。通过选择保存数据"另存为"。 注意:这些测试将包括多个值，但最重要的值是切片，这表明该系列中的时间点，长度，这表明该线（ 即星体微管）的长度。含有其它测量列（ 例如，区域，平均像素值，角度等 ）可以被保持或丢弃。 </li><li>创建通过右击并使用"插入"功能的电子表格的新列。输入每个时间点的时间（s）。 </li><li> 创建使用"插入线微管长度（μm）的作为时间（s）的函数的XY散点图图表"特征;选择的长度测量为‘Y’和时间列为‘X’。 <ol><li>通过寻找长度随着时间的推移从步骤7.10散点图正负变化确定聚合，解聚，和暂停的区域。 注:聚合事件由至少0.5μm的微管增加长度跨越至少3个时间点和适合的线性回归用的R 2≥0.9，R值&gt; 0（ 图1B和D，绿点）。解聚的事件跨至少3个时间点由至少0.5μm的微管减小长度和适合的线性回归用的R 2≥0.9和R值&lt;0（ 图1B和D，粉红色点）。暂停事件是从聚合和解聚分开。停顿被定义为小于0.5μm跨越至少3个时间点的微管长度的净变化;适合与-0.4和0.4之间的R值的线性回归;和具有斜面，是不是从零统计学差异，如通过F-检验确定。 </li><li>使用散点图为指导，确定的时间段时长的变化是积极的，并强调他们在一个绿色。突出的时区时的长度变化是在粉红色的彩色负片。 </li><li>计算各线性回归突出显示的部分，并记录R-和R 2 -值在列接近的区域每个部分。分类每个色区域，可以是聚合事件或解聚的事件。 </li></ol></li><li>除以长度的净变化通过改变在时间上对每个生长事件计算聚合率。记录这些结果列相邻的线性回归值从步骤7.10.3。 </li><li>除以长度的净变化通过改变在时间上对每个shrinkin计算解聚速率摹事件。记录这些结果列相邻于聚合速度的值从步骤7.11。 </li><li>通过所有的生长事件13的总时间除以聚合到解聚的转变的数目计算灾难频率。记录这些结果列相邻的解聚速率值从步骤7.12。 </li><li>通过所有收缩事件13的总时间除以解聚到聚合转变的数量计算出的救援频率。记录这些结果列相邻于灾难频率值从步骤7.13。 </li><li>除以由图像获取的总持续时间的所有长度的变化，在步骤7.10.1中计算出的绝对值的总和，并且通过27.0833相乘以获得每秒14微管蛋白亚单位计算动态性。记录相邻的救援频率VAL这些结果列从步骤7.14的UE并保存结果表。 注意:这是可以自动在使用定制的程序步骤7.10-7.15所描述的分析方法（Estrem，等人 ，手稿提交）。该方案旨在通过按照上述详细描述的参数，以确定在所述长度测量聚合和解聚的周期。 </li></ol>

<ol>
	<li>Zanic, M., Widlund, P. O., Hyman, A. A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nat Cell Biol. 15 (6), 688-693 (2013).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Curr Biol. 10 (14), 865-868 (2000).</li><li>Byers, B., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+spindles+and+spindle+plaques+in+the+cell+cycle+and+conjugation+of+Saccharomyces+cerevisiae.">Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.</a> J Bacteriol. 124 (1), 511-523 (1975).</li><li>Marschall, L. G., Jeng, R. L., Mulholland, J., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Tub4p,+a+yeast+gamma-tubulin-like+protein:+implications+for+microtubule-organizing+center+function.">Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.</a> J Cell Biol. 134 (2), 443-454 (1996).</li><li>Spang, A., Geissler, S., Grein, K., Schiebel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Tubulin-like+Tub4p+of+Saccharomyces+cerevisiae+is+associated+with+the+spindle+pole+body+substructures+that+organize+microtubules+and+is+required+for+mitotic+spindle+formation.">gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation.</a> J Cell Biol. 134 (2), 429-441 (1996).</li><li>Winey, M., Bloom, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+form+and+function.">Mitotic spindle form and function.</a> Genetics. 190 (4), 1197-1224 (2012).</li><li>Gupta, M. L. 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=beta-Tubulin+C354+mutations+that+severely+decrease+microtubule+dynamics+do+not+prevent+nuclear+migration+in+yeast.">beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.</a> Mol Biol Cell. 13 (8), 2919-2932 (2002).</li><li>Moore, J. K., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+mitosis+with+cell+polarity:+Molecular+motors+at+the+cell+cortex.">Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.</a> Semin Cell Dev Biol. 21 (3), 283-289 (2010).</li><li>Gartz Hanson, 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=Novel+alpha-tubulin+mutation+disrupts+neural+development+and+tubulin+proteostasis.">Novel alpha-tubulin mutation disrupts neural development and tubulin proteostasis.</a> Dev Biol. 409 (2), 406-419 (2016).</li><li>Fees, C. P., Aiken, J., O'Toole, E. T., Giddings, T. H. J., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+negatively+charged+carboxy-terminal+tail+of+beta-tubulin+promotes+proper+chromosome+segregation.">The negatively charged carboxy-terminal tail of beta-tubulin promotes proper chromosome segregation.</a> Mol Biol Cell. 27 (11), 1786-1796 (2016).</li><li>Song, S., Lee, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+of+Saccharomyces+cerevisiae+CDC5+in+cytokinesis.">A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.</a> J Cell Biol. 152 (3), 451-469 (2001).</li><li>Knop, 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=Epitope+tagging+of+yeast+genes+using+a+PCR-based+strategy:+more+tags+and+improved+practical+routines.">Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.</a> Yeast. 15 (10B), 963-972 (1999).</li><li>Kosco, K. 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=Control+of+microtubule+dynamics+by+Stu2p+is+essential+for+spindle+orientation+and+metaphase+chromosome+alignment+in+yeast.">Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.</a> Mol Biol Cell. 12 (9), 2870-2880 (2001).</li><li>Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+stabilization+of+microtubule+dynamic+instability+in+vitro+by+vinblastine.">Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine.</a> Biochemistry. 32 (5), 1285-1293 (1993).</li><li>Aiken, J., Sept, D., Costanzo, M., Boone, C., Cooper, J. A., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+novel+and+discrete+functions+for+tubulin+carboxy-terminal+tails.">Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.</a> Curr Biol. 24 (12), 1295-1303 (2014).</li><li>Straight, A. F., Marshall, W. F., Sedat, J. W., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+living+budding+yeast:+anaphase+A+but+no+metaphase+plate.">Mitosis in living budding yeast: anaphase A but no metaphase plate.</a> Science. 277 (5325), 574-578 (1997).</li><li>Khmelinskii, A., Lawrence, C., Roostalu, J., Schiebel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc14-regulated+midzone+assembly+controls+anaphase+B.">Cdc14-regulated midzone assembly controls anaphase B.</a> J Cell Biol. 177 (6), 981-993 (2007).</li><li>Markus, S. M., Omer, S., Baranowski, K., Lee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Plasmids+for+Fluorescent+Protein+Tagging+of+Microtubules+in+Saccharomyces+cerevisiae.">Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.</a> Traffic. 16 (7), 773-786 (2015).</li></ol>

作者宣称，他们没有竞争的经济利益。

芽殖酵母模型聚集在体内环境的微管动力学的高分辨率测量，包括对时间的能力，形象单一微管和操纵使用酵母遗传学的工具，微管蛋白和微管监管机构的能力提供了重要的优势。 所述涂覆的伴刀豆球蛋白室提供许多优于先前所描述的设备中，包含熔化的琼脂垫。与腔室玻片可以被预先制成并存储长期或可以立即使用。此外，该涂覆的伴刀豆球蛋白室能够培养的酵母?...

微管是由αβ微管蛋白的蛋白质亚基的细胞骨架的聚合物和在多种细胞环境中，包括胞内转运，细胞分裂，形态发生和运动性的被使用。要建立微管网络对于这些不同的角色，细胞必须仔细调节何时何微管形式。这种调节是通过控制反应完成，要么组装αβ微管蛋白亚基插入微管聚合物或拆卸的微管转化为游离亚基;这被称为微管动力学。 微管字段的一个主要目标是阐明调节微管动力学，包括结合于微管蛋白和/或微管的αβ微管蛋白亚单元和外在调节研究的分子机制。行之有效的实验方法是在体外用纯化的αβ微管蛋白的蛋白质在COM重建该系统中，常常bination用纯化的外在调节器。虽然这是一个有用的方法，很显然，在重构的系统微管动力学从活细胞中观察到的强烈不同。例如，微管生长更快和收缩体内比体外慢。这些差异可以归因于已知的外在调节器1的可用性，以及在细胞中尚未未定义的因素。因此，关键是要确定微管调节剂和破坏在天然细胞环境动力学突变体的活动。 虽然后生动物模型已经被证明是研究微管功能和高阶组织现行制度，有几个实际问题严重限制了这些模型的效用微管动力学的精确测量。首先，大量的微管，从几十到上千个细胞，使其难以理直气壮跟踪单个微管随着时间的推移。许多研究通过成像选择性定位于微管的端部，如在端部结合（EB）家族蛋白的蛋白应对这一挑战。然而，这些蛋白质是已知的唯一定位于在后生动物2生长微管的端部。因此，这些蛋白质的效用是有限的，以直接测量生长速率，而只有间接测量动力学的其他方面，诸如灾难的频率。其次，尽管在基因组编辑技术的进步，产生稳定表达荧光标记的微管蛋白或引入突变，以选择性地操纵微管蛋白的微管或调节细胞仍然是一个挑战显著。此外，许多微管蛋白同种型的后生动物存在混淆的突变如何影响个人微管蛋白基因的研究。 芽殖酵母系统提供几个重要的优点用于体内 microtubu测量乐动态。酵母具有简化的微管网络，它允许个别微管的可视化。在酵母中，微管从组织称为纺锤体极体（SPBS），其被嵌入在核膜3中心发出。所述SPBS用作支架用于该成核微管4,5γ微管蛋白小复合物。 SPBS核两类微管，纺锤体微管及微管星体的。纺锤体微管伸入和核质是重要的，用于经由微管的着丝点附连到染色体和用于经由重叠极间微管6稳定主轴。相比之下，微管星体向外项目进入细胞质，并比较少见相比纺锤体微管的密集网络。在有丝分裂，预后期细胞具有1-2只从任一SPB发出星体微管;这些都是普通的Individual微管，而不是作为束7。星体微管的有丝分裂过程中的作用是细胞核和主轴移动到母亲和芽的隔间，被称为芽颈部之间的交界处。这种运动包括良好定义的途径上星体微管生成的力，拉细胞核和主轴朝向并最终进入芽颈部8。 酵母系统的另一个优点是它的遗传学，其可用于研究的微管调节剂和微管蛋白亚单位具有无可比拟的精度的效用。酵母也具有微管蛋白同种型的简化剧目:单β微管蛋白基因（TUB2）和两个α微管蛋白基因（TUB1和TUB3）。突变可以容易地引入这些基因，从而以均匀的微管蛋白人口9,10研究。有许多的广泛对于标签的微管结构可用，而且这些设置可以针对在染色体位点稳定表达的整合（见讨论）。 该方法的总的目标是图像单个微管在活酵母细胞在四个维度（X，Y，Z和T​​）为微管动力学的高分辨率测量。用于荧光标记的微管蛋白在酵母细胞中的组成型，低水平表达整合构建体的方法进行说明。在成像之前，活细胞被安装到已经涂覆了凝集素伴刀豆球蛋白A，以稳定长期成像细胞的滑动室。用于图像采集的最佳参数，以及对数据进行分析的工作流，也有所说明。

<table><tbody><tr><td>DOB (dropout bases)</td><td>Sunrise science&amp;nbsp;</td><td>1650</td><td></td></tr><tr><td>CSM-Leu</td><td>Sunrise science&amp;nbsp;</td><td>1005</td><td></td></tr><tr><td>Agar</td><td>Ameresco</td><td>N833</td><td></td></tr><tr><td>100 mm polystyrene plates</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>ssDNA (Samon Sperm)&amp;nbsp; in sterile DiH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;D7656</td><td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Synthetic Complete Media&amp;nbsp;</td><td>Sunrise science&amp;nbsp;</td><td>1459-100</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L7647</td><td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>Microscope Coverslips</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td>paraffin film</td></tr><tr><td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td><td></td><td></td><td>melt at 75 &amp;ordm;C before use</td></tr><tr><td>forceps&amp;nbsp;</td><td>Fisher Scientific</td><td>16-100-106</td><td></td></tr><tr><td>Poyethylene glycol (PEG) 3350</td><td>Sigma-Aldrich&amp;nbsp;</td><td>202444</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ti E inverted Perfect Focus microscope</td><td>Nikon Instruments</td><td>MEA53100</td><td></td></tr><tr><td>1.45 NA 100X CFI Plan Apo objective</td><td>Nikon Instruments</td><td>MRD01905</td><td></td></tr><tr><td>Piezo electric stage&amp;nbsp;</td><td>Physik Instrumente</td><td>P-736</td><td></td></tr><tr><td>Spinning disk scanner</td><td>Yokogawa</td><td>CSU10</td><td></td></tr><tr><td>Laser combiner module</td><td>Agilent Technologies</td><td>MCL400B</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor Technology</td><td>iXon Ultra 897&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS Elements software</td><td>Nikon Instruments</td><td>MQS31100</td><td></td></tr><tr><td>Microsoft Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks, Inc</td><td></td><td></td></tr><tr><td>ImageJ64</td><td>NIH</td><td></td><td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td></tr><tr><td>Bio-Formats Importer plug-in</td><td>Open Microscopy Environment</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pUC19-LEU2::GFP-TUB1</td><td></td><td>pSK1050</td><td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td></tr></tbody></table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

动态微管是许多细胞过程的根本，微管动力学的精确测量可以提供深入了解细胞如何调节这些过程和突变的影响如何调控基因。微管动力学的后生动物模型的量化有许多相关的挑战，包括高密度微管和限制遗传操作。相比之下，芽殖酵母模型提供了克服这些挑战的优势。这个协议描述来测量在活酵母细胞单个微管的动力学的方法。表达荧光标记的微管蛋白的细胞粘附到组装的滑动室，从而允许稳定的隔时图像采集。用于高速的详细指南，四维图像采集本发明还提供，以及用于在共焦图像栈量化动态微管的性质的协议。该方法中，结合常规酵母遗传学省IDES了一种方法，是唯一适合用于定量评估该改变微管蛋白亚单位的活性的微管调节剂或突变的影响。

在活酵母细胞测量微管动力学提供了令人信服的工具，以评估在编码微管调节剂或微管蛋白基因中的突变如何亚基冲击聚合和解聚率，以及这些状态之间的过渡频率。 图1显示在野生型细胞中的时间序列的星体微管动力学的和β微管蛋白具有突变的突变型细胞（tub2-430Δ）。微管标记有GFP标记的α微管蛋白可视化微管的长度。红色箭头跟踪每个图像?...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

出芽酵母是研究体内微管动力学由于其强大的遗传学和其微管骨架的简单性的有利的模型。以下方案描述了如何变换和培养的酵母细胞，获得共焦显微镜图像，和定量分析在活酵母细胞的微管动力学。

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

high-resolution-imaging-analysis-individual-astral-microtubule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.2K 次观看.  University of Colorado School of Medicine. 出芽酵母是研究体内微管动力学由于其强大的遗传学和其微管骨架的简单性的有利的模型。以下方案描述了如何变换和培养的酵母细胞，获得共焦显微镜图像，和定量分析在活酵母细胞的微管动力学。 

视频: High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

科研

生物学

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

生物化学

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

基本的有丝分裂纺锤体的重组在球形乳液液滴

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

高分辨率成像和芽殖酵母个人星体微管动力学分析

摘要

探索更多视频

此视频中的章节

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

裂殖酵母性生命周期的显微镜

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

基本的有丝分裂纺锤体的重组在球形乳液液滴

在裂殖酵母中Cytokinetic活动的时空分析

酵母的4D显微镜

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

亚细胞结构的超分辨率活细胞成像

高分辨率成像和芽殖酵母个人星体微管动力学分析

摘要

探索更多视频

此视频中的章节

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

裂殖酵母性生命周期的显微镜

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

基本的有丝分裂纺锤体的重组在球形乳液液滴

在裂殖酵母中Cytokinetic活动的时空分析

酵母的4D显微镜

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

亚细胞结构的超分辨率活细胞成像

通过TIRF显微镜在体外同时可视化交联和单微管的动力学