This work was supported by startup funds from The University of Tennessee and TN-SCORE, a multi-disciplinary research program sponsored by NSF-EPSCoR (EPS-1004083).

1.样品的制备<ol><li>生长表达纺锤体极体标记Sad1在每个-mCherry 23和II型肌球蛋白调节轻链RLC1-番茄24中，YE液体培养基在32℃下8代裂殖酵母细胞。 注:对于温度敏感的突变体，在25℃生长细胞。生长细胞对数中期，以叶为0.5的OD 600。欲了解更多有关裂殖酵母的生长条件是指裂殖酵母实验手册25。 </li><li>豫备（或最少）的媒体用0.6％琼脂糖（琼脂糖的媒体表现出相较于琼脂少荧光背景）。到熔融的介质添加的抗坏血酸为1mM（维生素C）。维生素C的骤冷，由于被释放到光漂白，从而减少光毒性自由基。 </li><li>倒入3 - 维生素C的股价融化到一个玻璃底培养皿媒体4毫升。让媒体冷却和固化。 注意:在培养皿的玻璃的厚度取决于适于在显微镜盖玻片的厚度。在这里，使用盖玻片号1.5。 </li><li>轻轻地从用锋利的解剖刀的帮助培养皿提高固化介质。放置介质板和培养皿为了防止板坯从落回到菜（ 图1）之间的枪头。 </li><li>取1毫升新鲜生长的细胞如上面1.1中描述。轻轻旋转细胞在300 XG 30秒。弃去上清液。悬浮细胞在弃去上清后仍保留在管中的少量残余介质。 </li><li>负载2 - 5μL媒体板和培养皿的玻璃底部之间重悬浮细胞培养。细胞应该在玻璃上。放置媒体和盘之间的枪头使细胞培养物的容易装载。 </li><li>轻轻地除去枪头和放置媒体板坯回其原来的位置上的铜lture菜。请一定要避免任何气泡。如果需要通过滑动枪头在介质板顶部的背面轻轻压出气泡。 </li><li>让细胞坐在在该镜将被执行的温度培养皿30分钟至一小时在黑暗中。由于对细胞生长条件改变这一点很重要，以防止任何冲击。 </li></ol> 2.图像采集<ol><li>放置的油上的培养皿的玻璃的外侧的下降。放在倒置显微镜培养皿。 </li><li>为了最大限度地减少叶媒体的自动荧光使用GFP滤波器具有窄波长范围（520 - 535纳米）。 </li><li>专注于细胞的中间平面，并确保他们是好间隔并没有太拥挤。确保以启动图像采集的细胞在细胞周期的适当阶段。在这个实验中，遵循后期G2细胞。 </li><li> 程序中的图像采集软件进行tO拍摄GFP，RFP和细胞的DIC图像。 <ol><li>此实验使用10毫秒的曝光时间为DIC和75毫秒的曝光时间的GFP和RFP滤波器。曝光时间依赖于显微镜，所研究的蛋白质，实验的持续时间的设置。对于给定的显微镜设置，使用50％的激光功率。 </li><li>要在3个维度，程序中的图像采集软件，Z-级成像捕捉图像。使用0.4μm的周围的小区的中央聚焦点步长大小为3.0微米（半总的z）的距离。这导致了每个时间点16的Z-帧为6微米的总Z方向的距离捕获。 注:Z系列图像采集允许主轴极体的捕获。 </li></ol></li><li>取的图像，每2分钟。根据实验的需要改变曝光时间。曝光时间也可以根据所关注和信号强度的蛋白质变化。但是请注意，短呃因此细胞只能在短时间内跟随间隔或更长的曝光时间的增加光毒性由于漂白和。 </li><li>获取图像，直到细胞由DIC图像观察到物理上独立的，或者软件90分钟后停止收购方案。 </li></ol> 3.图像分析<ol><li> cytokinetic事件的时空分析 <ol><li>使用图像采集软件使在Z帧的每个时间点的预测。使不同的文件为所获取的图像的每一个波长。 </li><li>导出此投影时间点系列作为.tiff文件。 </li><li>分析使用ImageJ软件图像。打开波长中的任一项的图像系列。 </li><li>选择的小区来进行研究。双击工具栏的行选项。这将打开另一个窗口调整线条宽度。调节线宽度，以对应于单元宽度。对于WT细胞，这是大约40 - 50像素。绘制沿一条线感兴趣的细胞的长轴。 </li><li>点击分析&gt;工具&gt;投资回报率Manager，然后单击添加。这将在稍后选定的行添加到ROI窗口使用。请不要关闭此窗口。 </li><li>点击感兴趣的图像，并选择编辑&gt;选择&gt;拉直。这将打开另一扇窗。检查"处理整个堆栈"。这将理顺水平的细胞。 </li><li>点击图片&gt;变换&gt;旋转90度右。此图片现在垂直拉直。 </li><li>点击图片&gt;栈&gt;制作蒙太奇。这将打开一个窗口分配行和列的数目为堆栈，用于图像大小的比例因子，对蒙太奇的第一和最后的片（帧）和用于蒙太奇帧增量。检查标签的切片，然后回车。 </li><li>如所示与所关注的细胞的一蒙太奇的窗口，打开的其他波长的图像系列。 </li><li>点击ROI的经理行标识符。这将选择对第二图像文件相同的细胞。重复步骤3.1.6 - 3.1.8。这将打开第二图像的一个蒙太奇。 </li><li>在与主轴极体标记Sad1在-mCherry形象，寻找主轴极体标记的分离发生和标记该时间点为时间0。 </li><li>按照图像上的RLC1番茄信号随着时间的推移，寻找，而不是RLC1番茄的补丁出现作为一个独特的线上的信号。这个时间点，标志着肌动球蛋白环组件/成熟阶段开始的完成。 </li><li>通过电影下一个滚动一段时间，以确定何时RLC1-番茄环开始的尺寸减小。此标记为成熟期/发病环收缩的结束。 </li><li>按照随着时间的推移RLC1-番茄信号整个收缩直到它出现在细胞轴的中间的点。此标记为隔膜进入的环收缩/结束的结束。 </li><li>继DIC图像的蒙太奇，确定最终的细胞分离。记录的时间点的细胞的物理分离。 </li><li>对于纺锤体极体分离使用ImageJ的直线工具测量随着时间的推移在两个主轴极体之间的距离。剧情随着时间的推移主轴极体之间的距离。 </li><li>记录不同时间点的不同cytokinetic事件来计算每个cytokinetic阶段的持续时间和与纺锤极体的分离随着时间的推移进行比较。 </li></ol></li><li> cytokinetic事件的空间分析 <ol><li>选择带有明显的肌动球蛋白环的细胞。双击ImageJ的工具栏的行选项。这将打开另一个窗口调整线条宽度。调节线宽度，以对应于所述环的厚度（15 - 20个像素）。绘制沿环照顾，以确保环的整个厚度的线是包括在内。 </li><li>点击分析&gt;工具&gt;投资回报率Manager，然后单击添加。这将在稍后选定的行添加到ROI窗口使用。请不要关闭此窗口。 </li><li>点击感兴趣的图像，并选择编辑&gt;选择&gt;拉直。这将打开另一扇窗。检查"处理整个堆栈"。这将理顺水平环。 </li><li>使用图像复位最亮面的强度在拉直Z堆栈（从3.1.6）&gt;调整&gt;亮度/对比度&gt;重置。 </li><li>单击该选项卡STK&gt; 3D项目上。这将打开一个对话框。改变投射方法亮的点和切片间距为2或3个像素。最后，旋转X或Y轴的变化轴（这将取决于所需的视角改变），单击插值并单击确定以生成图像的3D投影。使用滚动条下方的形象旋转图像。 </li><li>打开使用ImageJ其它波长的图像系列。 </li><li>点击ROI的经理行标识符。这将选择第二图像文件相同的环。重复步骤4.2.4和4.2.5。这将与其他波长的图像中打开一个三维环。 </li><li>随着这两种3D图像环打开时，单击图像&gt;彩色&gt;合并通道。这将打开一个盒子。选择从下拉的期望相应的颜色菜单每一个图像，然后单击确定。这将打开两个在不同波长的图像的组合。 </li><li>与在cytokinetic环问候本地化或ingressing膜比较不同的标记的本地化。 RLC1-GFP是用于cytokinetic环的标记。蛋白质共同本地化与RLC1-GFP定位于环，而蛋白质本地化背后RLC1-GFP信号定位于ingressing膜。 注:感兴趣的蛋白质必须是不同的荧光团相比，该环标记的。 </li><li>生成期间胞质的不同阶段3维环全部通过胞质来比较蛋白质的空间定位。 </li></ol></li><li> 蛋白质DISTRI分析bution在cytokinetic环<ol><li>为了比较和分析蛋白质的分布沿环形衡量合作效率蛋白质分布的方差。高共高效方差指示沿分裂部位的蛋白质的分布不均，并建议不当环组件，成熟或隔膜形成，这取决于相位成像或分析。 </li><li>使用3维重构cytokinetic环（来自3.2.2），将图像分为至少4个象限，使得环的四分之一出现在每个象限中。对于这种用直线工具画在图像上垂直线。每一行点击图片后&gt;覆盖&gt;添加选择或使用短切按Ctrl + B。 </li><li>进入分析&gt;设置测量。选择在打开的对话框平均灰度值。点击OK。 </li><li>使用上面作为指南绘制一个矩形使用矩形工具来定义每个象限绘制的线条。 </li><li>为了测量每个象限，点击的平均强度在分析&gt;测量或使用快捷键Ctrl + M。 </li><li>记录所有四个象限平均灰度值（MG 1 -MG 4）。 </li><li>选择在环作为背景选择以外的每个象限一个小区域。 </li><li>衡量背景选择为所有四个象限平均灰度值（BG 1 -BG 4）。 </li><li> LI&gt;背景扣除每个象限使用MG式- -平均1（BG 1:BG 4）以下=环的强度在每一个象限（IRQ）。在此阶段有4个的IRQ值，每个象限。 </li><li>下一个计算方差的系数，使用下列公式标准偏差每个环（IRQ 1:IRQ 4）/平均值（IRQ 1:IRQ 4）。计算平均共同有效方差每个菌株，并比较所述其他菌株。 注:在共同有效方差的统计学显著增加的菌株与对照相比INDicates不均匀分布的蛋白质/沿着分工站点胞质那个阶段交付。 </li></ol></li></ol>

<ol>
	<li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cytokinesis+in+eukaryotes.">Mechanics of cytokinesis in eukaryotes.</a> Curr Opin Cell Biol. 22 (1), 50-56 (2010).</li><li>Guertin, D. A., Trautmann, S., McCollum, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis+in+eukaryotes.">Cytokinesis in eukaryotes.</a> Microbiol Mol Biol Rev. 66 (2), 155-178 (2002).</li><li>Xu, X., Vogel, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+protein+promotes+cleavage+furrow+maturation+during+cytokinesis.">A secreted protein promotes cleavage furrow maturation during cytokinesis.</a> Curr Biol. 21 (2), 114-119 (2011).</li><li>Sagona, A. P., Stenmark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis+and+cancer.">Cytokinesis and cancer.</a> FEBS Lett. 584 (12), 2652-2661 (2010).</li><li>Fujiwara, T., 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=Cytokinesis+failure+generating+tetraploids+promotes+tumorigenesis+in+p53-null+cells.">Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.</a> Nature. 437 (7061), 1043-1047 (2005).</li><li>Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis+in+development+and+disease:+variations+on+a+common+theme.">Cytokinesis in development and disease: variations on a common theme.</a> Cell Mol Life Sci. 64 (23), 3044-3058 (2007).</li><li>Daniels, M. J., Wang, Y., Lee, M., Venkitaraman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+cytokinesis+in+cells+deficient+in+the+breast+cancer+susceptibility+protein+BRCA2.">Abnormal cytokinesis in cells deficient in the breast cancer susceptibility protein BRCA2.</a> Science. 306 (5697), 876-879 (2004).</li><li>Storchova, Z., 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=From+polyploidy+to+aneuploidy,+genome+instability+and+cancer.">From polyploidy to aneuploidy, genome instability and cancer.</a> Nat Rev Mol Cell Biol. 5 (1), 45-54 (2004).</li><li>Lee, I. J., Coffman, V. C., Wu, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile-ring+assembly+in+fission+yeast+cytokinesis:+Recent+advances+and+new+perspectives.">Contractile-ring assembly in fission yeast cytokinesis: Recent advances and new perspectives.</a> Cytoskeleton (Hoboken). 69 (10), 751-763 (2012).</li><li>Balasubramanian, M. K., Bi, E., Glotzer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+cytokinesis+in+budding+yeast,+fission+yeast+and+animal+cells.">Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells.</a> Curr Biol. 14 (18), R806-R818 (2004).</li><li>Proctor, S. A., Minc, N., Boudaoud, A., Chang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+turgor+pressure,+the+contractile+ring,+and+septum+assembly+to+forces+in+cytokinesis+in+fission+yeast.">Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast.</a> Curr Biol. 22 (17), 1601-1608 (2012).</li><li>Munoz, 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=Extracellular+cell+wall+beta(1,3)glucan+is+required+to+couple+septation+to+actomyosin+ring+contraction.">Extracellular cell wall beta(1,3)glucan is required to couple septation to actomyosin ring contraction.</a> J Cell Biol. 203 (2), 265-282 (2013).</li><li>Wang, N., Lee, I. J., Rask, G., Wu, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+TRAPP-II+Complex+and+the+Exocyst+in+Membrane+Deposition+during+Fission+Yeast+Cytokinesis.">Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis.</a> PLoS Biol. 14 (4), e1002437 (2016).</li><li>Liu, J., Wang, H., McCollum, D., Balasubramanian, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drc1p/Cps1p,+a+1,3-beta-glucan+synthase+subunit,+is+essential+for+division+septum+assembly+in+Schizosaccharomyces+pombe.">Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.</a> Genetics. 153 (3), 1193-1203 (1999).</li><li>Cortes, J. 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=The+(1,3)beta-D-glucan+synthase+subunit+Bgs1p+is+responsible+for+the+fission+yeast+primary+septum+formation.">The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation.</a> Mol Microbiol. 65 (1), 201-217 (2007).</li><li>Cortes, J. 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=Cooperation+between+Paxillin-like+Protein+Pxl1+and+Glucan+Synthase+Bgs1+Is+Essential+for+Actomyosin+Ring+Stability+and+Septum+Formation+in+Fission+Yeast.">Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast.</a> PLoS Genet. 11 (7), e1005358 (2015).</li><li>Cortes, J. C., Ishiguro, J., Duran, A., Ribas, 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=Localization+of+the+(1,3)beta-D-glucan+synthase+catalytic+subunit+homologue+Bgs1p/Cps1p+from+fission+yeast+suggests+that+it+is+involved+in+septation,+polarized+growth,+mating,+spore+wall+formation+and+spore+germination.">Localization of the (1,3)beta-D-glucan synthase catalytic subunit homologue Bgs1p/Cps1p from fission yeast suggests that it is involved in septation, polarized growth, mating, spore wall formation and spore germination.</a> J Cell Sci. 115 (Pt 21), 4081-4096 (2002).</li><li>Liu, J., Tang, X., Wang, H., Oliferenko, S., Balasubramanian, M. 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+localization+of+the+integral+membrane+protein+Cps1p+to+the+cell+division+site+is+dependent+on+the+actomyosin+ring+and+the+septation-inducing+network+in+Schizosaccharomyces+pombe.">The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe.</a> Mol Biol Cell. 13 (3), 989-1000 (2002).</li><li>Royou, A., Field, C., Sisson, J. C., Sullivan, W., Karess, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reassessing+the+role+and+dynamics+of+nonmuscle+myosin+II+during+furrow+formation+in+early+Drosophila+embryos.">Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos.</a> Mol Biol Cell. 15 (2), 838-850 (2004).</li><li>Figard, L., Xu, H., Garcia, H. G., Golding, I., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasma+membrane+flattens+out+to+fuel+cell-surface+growth+during+Drosophila+cellularization.">The plasma membrane flattens out to fuel cell-surface growth during Drosophila cellularization.</a> Dev Cell. 27 (6), 648-655 (2013).</li><li>Wei, 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=Unique+Spatiotemporal+Activation+Pattern+of+Cdc42+by+Gef1+and+Scd1+Promotes+Different+Events+during+Cytokinesis.">Unique Spatiotemporal Activation Pattern of Cdc42 by Gef1 and Scd1 Promotes Different Events during Cytokinesis.</a> Mol Biol Cell. , (2016).</li><li>Nabeshima, K., 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=Dynamics+of+centromeres+during+metaphase-anaphase+transition+in+fission+yeast:+Dis1+is+implicated+in+force+balance+in+metaphase+bipolar+spindle.">Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle.</a> Mol Biol Cell. 9 (11), 3211-3225 (1998).</li><li>Johnson, A. E., Gould, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dma1+ubiquitinates+the+SIN+scaffold,+Sid4,+to+impede+the+mitotic+localization+of+Plo1+kinase.">Dma1 ubiquitinates the SIN scaffold, Sid4, to impede the mitotic localization of Plo1 kinase.</a> EMBO J. 30 (2), 341-354 (2011).</li><li>Yonetani, A., Chang, F. <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+cytokinesis+by+the+formin+cdc12p.">Regulation of cytokinesis by the formin cdc12p.</a> Curr Biol. 20 (6), 561-566 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fission Yeast: A laboratory Manual. , (2016).</li><li>Hachet, O., Simanis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mid1p/anillin+and+the+septation+initiation+network+orchestrate+contractile+ring+assembly+for+cytokinesis.">Mid1p/anillin and the septation initiation network orchestrate contractile ring assembly for cytokinesis.</a> Genes Dev. 22 (22), 3205-3216 (2008).</li><li>Zhou, Z., 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+contractile+ring+coordinates+curvature-dependent+septum+assembly+during+fission+yeast+cytokinesis.">The contractile ring coordinates curvature-dependent septum assembly during fission yeast cytokinesis.</a> Mol Biol Cell. 26 (1), 78-90 (2015).</li><li>Chen, B. 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=Lattice+light-sheet+microscopy:+imaging+molecules+to+embryos+at+high+spatiotemporal+resolution.">Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.</a> Science. 346 (6208), 1257998 (2014).</li><li>Huang, Y. S., Karashima, T., Yamamoto, M., Hamaguchi, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular-level+investigation+of+the+structure,+transformation,+and+bioactivity+of+single+living+fission+yeast+cells+by+time-+and+space-resolved+Raman+spectroscopy.">Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy.</a> Biochemistry. 44 (30), 10009-10019 (2005).</li><li>Huang, C. K., Ando, M., Hamaguchi, H. O., Shigeto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disentangling+dynamic+changes+of+multiple+cellular+components+during+the+yeast+cell+cycle+by+in+vivo+multivariate+Raman+imaging.">Disentangling dynamic changes of multiple cellular components during the yeast cell cycle by in vivo multivariate Raman imaging.</a> Anal Chem. 84 (13), 5661-5668 (2012).</li><li>Le Goff, X., Woollard, A., Simanis, V. <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+the+cps1+gene+provides+evidence+for+a+septation+checkpoint+in+Schizosaccharomyces+pombe.">Analysis of the cps1 gene provides evidence for a septation checkpoint in Schizosaccharomyces pombe.</a> Mol Gen Genet. 262 (1), 163-172 (1999).</li></ol>

作者宣称没有竞争经济利益。

在这里，我们已经描述了协议来研究在裂殖酵母cytokinetic事件以时间的方式。这里所描述的协议提供了参照彼此不同cytokinetic事件的时间分辨率;蛋白质招聘或参照cytokinetic缺相的时间;整个胞质的不同阶段的环的结构;和胞质的参照的有丝分裂的进展。与此协议是可能的精确定义可以在不同的突变体而改变，从而提供涉及不同cytokinetic相蛋白质的更详细的了解cytokinetic相。另外，为了在时间上区分不同cytokinetic阶段的能力将使导致不同cytokinetic相的组织的分子机制的分析。不同cytokinetic事件的时间分辨率也使cytokinetic环结构和蛋白质分布的分析贯穿不同的事件。不同的蛋白质的时空定位可以比较并用这种方法进行分析。通过使用适宜的荧光团，多种蛋白质，可以研究和相互比较。除了这里，我们还描述了如何沿环蛋白的分布可以被分析，以确定在除法站点蛋白质组织或交付。而这里描述的协议适用于裂殖酵母胞质，这种方法也可以被用来研究在其他酵母胞质和真菌与生长和成像条件的适当变化。 该协议涉及成像活细胞分裂他们。使用该协议成象的细胞需要最佳条件下生长，以避免任何pleotropic影响。应注意到不拥挤的细胞中的视场，如过度的细胞可导致迅速养分流失。另外在细胞中的荧光成像导致毒性由于免费自由基释放光致漂白的结果。这可以通过加入游离基淬火化合物如抗坏血酸被最小化。加入抗坏血酸允许在显微镜下的细胞的延长的成像。期间的图像采集，需要被成像以确保主轴杆机构的捕获的细胞的整个Z轴。主轴极体缺乏适当信号将不允许胞质适当时间分辨率。特别是，应小心精确地图像纺锤极体标记物的初始分离，这是时间0沿Z轴的细胞的适当的捕获也是环和隔垫的正确三维重建的关键。 图像圈和隔垫的横截面，这里描述的方案可以与微加工以及允许棒状裂殖酵母细胞是地方垂直成像沿CE的长轴改性LL作为已经在这里27日报道。虽然这种协议可以非常精确地捕获蛋白招募和定位随时间在细胞分裂位点的图像的空间分辨率是有限的，由于所使用的显微镜​​的分辨率。超分辨率显微镜可以帮助提高空间分辨率，但可能会影响时间分辨率。在显微镜如点阵灯表镜的最新进展可能有助于改善空间分辨率而不影响时间分辨率28。除了光镜，胞质裂殖酵母也可以使用拉曼光谱29,30研究随着时间的推移。 使用这种方法，我们报道了小GTP酶Cdc42的胞质21期间经历了独特的时空激活模式。这种方法使我们能够研究不同cytokinetic活动组织结束了时间和描述了这些事件的分子细节。报告表明，单独的组装肌动球蛋白环的存在是不足够用于有效膜开沟和胞质11，21，31。这里所描述的协议可以用于研究导致正确胞质肌动球蛋白环组件后的分子事件。此外，肌动球蛋白环及其对膜开沟和隔膜侵入效果的完整性进行检查。这将提供不同的分子事件共同导致成功分离更深入的了解。

胞质，在细胞分裂的最后一步，是适当的分化，发育，和生物体的存活一个复杂的过程是必不可少的。胞质分裂涉及到的组织，以确保成功细胞分离，同时保持基因组的完整性1多个事件。胞质涉及事件，其中一次组装的肌动球蛋白环经历收缩，这是并发膜膨胀和开沟，和细胞外细胞基质重塑，最后接着细胞脱落1，2，3。 cytokinetic事件的不当组织可导致细胞的分离和倍性的缺陷，并可能导致的疾病，如癌症4，5，6，7，8。的基本原则，使Òcytokinetic事件rganization都不能很好地理解，因此导致路障中的这些疾病的病因学的理解。 裂殖酵母粟酒裂殖酵母 （ 裂殖酵母）是研究胞质优异的模型系统，由于涉及1蛋白质的保守性质。裂殖酵母，肌动球蛋白环组件后，该环进入成熟/停留期，此时它不收缩9。成熟与肌动球蛋白环收缩，并发膜开沟和隔侵入开始结束。多年来开创性的工作给了我们肌动球蛋白环组件的一个相当不错的理解裂殖酵母1,9，10。在一些真核生物，包括裂殖酵母中，肌动球蛋白环的成功组装不足以膜开沟。 f中ission酵母，环缩单独不提供足够的力来克服对沟形成11内部膨压。最近的模型表明，这股力量代替由隔侵入11提供。在另一个模型中，质膜的扩展的作用已建议向沟形成12，13。环收缩和膜开沟不允许BGS1 / CPS1温度敏感突变cps1-191，对于主隔膜形成14，15中的主要酶发生。缺乏BGS1细胞显示缺陷主要隔，延长环收缩15，16。 BGS1是肌动球蛋白环组件17,18后成熟过程中招募到细胞分裂站点隔侵入。 SimilarlY，在果蝇胚胎细胞化过程中，环形缩窄是双相有显著缓慢的初始速度收缩19，类似在裂殖酵母中观察到的成熟阶段。双相环收缩可能会减慢膜开沟以确保足够的膜扩张20和细胞外基质的修改。这表明，肌动球蛋白环组件后，发生环收缩有效地仅当电池已经满足了沟形成的要求。它不能很好地理解，需要什么样的条件对于环组件后肌动球蛋白环收缩，也不是调节该过程的分子事件。我们最近表明，以下肌动球蛋白环组件中的小GTP酶的Cdc42经历一种独特的时空激活图案21。此模式是由Cdc42的胍核苷酸交换因子的独特的定位图案（建立全环基金）的激活Cdc42的。全球环境基金GEF1定位于组装肌动球蛋白环和促进环收缩和隔侵入发病，而SCD1定位于开沟膜和促进正常的隔膜形成。我们发现，通过其环基金建立的Cdc42的激活模式导致不同cytokinetic事件的调节。 理解的事件，最终导致细胞分离以下肌动球蛋白环组件的分子机制，需要按照时间和空间上的不同cytokinetic事件。裂殖酵母，胞质分裂首先涉及细胞核周围前驱节点，最终招II型肌球蛋白，所述formin CDC12，以及肌动球蛋白环组件所需的其他蛋白质的组装。到时的不同cytokinetic事件和提供一个参考帧，的纺锤体极体标志物（纺锤体形成）分离被认为是时间0 22。个的组件È肌动球蛋白环可通过监测在一段时间，如II型肌球蛋白调节轻链RLC1一个荧光标记的肌动球蛋白环蛋白的强度。在这里，我们描述了微观的方法随时间推移分析胞质的不同阶段。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast extract media</td>
			<td>Sunrise Science Products</td>
			<td>YES 225</td>
			<td>0.5% w/v yeast extract, 3% w/v glucose, 225mg/L adenine, histidine, leucine, uracil, and lysine</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem LE agarose, Lonza</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottomed culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Coverslip No. 1.5 was used. This will vary as per the microscope specifications used.&#160;</td>
		</tr>
		<tr>
			<td>VT-Hawk 2D array laser scanning confocal microscopy system</td>
			<td>Visitech International, UK</td>
			<td></td>
			<td>with an Olympus IX-83 inverted microscope with a 100X / numerical aperture 1.49 UAPO lens (Olympus) and EM-CCD digital camera (Hamamatsu).&#160;</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

spatiotemporal analysis of cytokinetic events in fission yeast

胞质，在细胞分裂的最后一步是维持基因组完整性的关键。正确的胞质分裂是细胞分化和发育非常重要。胞质分裂涉及一系列在时间上和空间上很好地协调活动。胞质涉及在分割部位的肌动球蛋白环的形成，随后通过环收缩，膜沟的形成和细胞外基质重塑。裂殖酵母， 裂殖酵母 （ 裂殖酵母）是揭示有大量的清晰度在胞质分裂的初始事件充分研究的模型系统。但是，我们不明白cytokinetic事件显然有什么不同spatiotemporally协调。为了确定这一点，需要分析很详细的不同cytokinetic事件在时间和空间。在这里，我们描述了一个显微镜的方法来研究不同cytokinetic EV需求测试的活细胞。使用这种方法，可以不同的时间cytokinetic事件和确定胞质中不同蛋白质的募集时间。此外，我们描述的协议在细胞分裂的部位比较蛋白质的定位和分布。这是一个基本的协议来研究胞质裂殖酵母，也可以用于其它酵母和真菌的系统。

表达该环标记裂殖酵母细胞，RLC1-GFP（绿色， 图2）和纺锤体极体标记Sad1在每个-mCherry（红， 图2）胞质中被成像。纺锤体极体标记的发病（白色箭头， 图2A，B），被认为是时间0 RLC1-GFP信号出现时间-4分钟参照纺锤极体的分离（红色箭头，图2A，2B）。 RLC1-GFP信号形成连续的环10分钟后纺锤极体的分离（空箭头， 图2A，B）的标记肌动球蛋白环组件的端部。肌动球蛋白环（RLC1-GFP）的启动环组件的完成和纺锤体极体分离后32分钟后在宽度22分钟，以降低（封闭箭头， 图2A，B）。环收缩结束收缩开始后20分钟，因为环组件的发病以来主轴极体分离52分钟，42分钟（白阿斯特里斯K， 图2A，B）。最后，细胞脱落发生72分钟后纺锤体极体分离（红色箭头， 图2A，B）。 环标记RLC1番茄和鼻中隔膜标记BGS1-GFP的定位在整个胞质分裂的网站进行了分析。环的三维重建透露，标有RLC1番茄肌动球蛋白环BGS1-GFP招募（ 图3，10分钟）之前组装。在环成熟阶段BGS1-GFP被招募到分割点和与肌动球蛋白环重叠如由RLC1-番茄（ 图3，30分钟）。这表明BGS1定位于招聘时的环。作为环收缩，BGS1-GFP也定位于相邻的收缩环（ 图3，40分钟）的ingressing膜。在收缩的结束时，BGS1-GFP信号是在侵入的膜的Barri可见器（ 图3，50分钟）。最后，收缩后RLC1-番茄信号不存在，而BGS1-GFP显示整个带圆盘状的外观（ 图3，60分钟）的膜屏障本地化。因此，这些观察结果表明，该隔膜合成酶BGS1定位于环组装后并在膜阻挡环收缩后仍然为17之前已经报道。 分析沿肌动球蛋白环蛋白质的分布，以确定cytokinetic事件（ 图4）的效率。沿着肌动球蛋白环蛋白或不均匀分布的非齐次已与受损的信令和低效cytokinetic环组件26相关联。这里，我们表明在成熟阶段两个3D重建环，表达的F栏蛋白Cdc15-GFP（ 图4）。图片在左侧示出了甚至Cdc15-GFP的分布沿环方差（0.098）系数低，而在右边的图像示出了分布不均方差系数高（0.226， 图 4）。 <img alt="图1" src="/files/ftp_upload/55109/55109fig1.jpg" /> 图1:培养皿成像的制备。细胞的原理，描述接种入玻璃底培养皿覆盖有媒体垫。详情请参见（第1节）协议。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55109/55109fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55109/55109fig2.jpg" /> 图2中的时序裂殖酵母 Cytokinetic事件。细胞表达SPINDL的（A）蒙太奇e极身体标记Sad1在-mCherry（左图），环蛋白RLC1-GFP（中图）和明场（右图）接受胞质显示在2分钟的间隔。白色箭头在17分钟标志着纺锤体极体（中心体）分离，红色箭头在27分钟标志着在师现场RLC1-GFP的初始招聘，开箭头标记环成熟，箭头在47分钟，星号标记，标志着环收缩发作在69分钟和红色箭头环收缩的结束在97分钟标志着细胞脱落。参照有丝分裂cytokinetic事件（B）的时间轴由纺锤极体的形成和分离为确定。细胞在25℃下成像。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55109/55109fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55109/55109fig3.jpg" /> 图3:CELL司网站在胞质分裂的不同阶段。肌动球蛋白环Z-栈的三维重建过程中细胞分裂的不同阶段为同一小区，环组件（10分钟后主轴极体分离），环成熟（30分钟后主轴极体分离），环收缩（40分钟后锭杆体分离），收缩的结束（50分钟后主轴极体分离），隔屏障（60分钟后主轴极体分离）。 RLC1番茄标志着环而BGS1-GFP标志着质膜包围隔。比例尺=5μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55109/55109fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55109/55109fig4.jpg" /> 图4:沿环蛋白的分布。三维图像重建肌动球蛋白环从野生C型表达Cdc15-GFP厄尔分为四个象限。共同有效每个环的方差中指出。比例尺=5μm以下。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55109/55109fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast at JoVE.com

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

裂殖酵母， 粟酒裂殖酵母是研究胞质极好的模型系统，在细胞分裂的最后阶段。在这里，我们描述了一个显微镜的方法来分析活裂殖酵母细胞不同cytokinetic事件。

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 ...

spatiotemporal-analysis-of-cytokinetic-events-in-fission-yeast

Nanyang Technological University

Research

JoVE Journal

Biology

7.3K 次观看.  University of Tennessee. 裂殖酵母， 粟酒裂殖酵母是研究胞质极好的模型系统，在细胞分裂的最后阶段。在这里，我们描述了一个显微镜的方法来分析活裂殖酵母细胞不同cytokinetic事件。 

视频: Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

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 ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

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

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

活细胞成像早期自噬活动:Omegasomes与超越

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

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

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 ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

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

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

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 ...

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 ...