We like to thank the members of the Dogterom lab for valuable discussions. We acknowledge Stefan Diez and Marcel Janson for reagents, and Samara Reck-Peterson for help with the purification of dynein. This work was supported by funding from the European Research Council (ERC Synergy grant: MODEL-CELL).

1.微流控芯片的制备注:对于主轴组件的自下而上的研究中，使用球形水包油乳液液滴。这些是由磷脂和表面活性剂的单层从周围油相分离的水的微滴。这些乳液液滴的微流体聚二甲基硅氧烷（PDMS）芯片内产生的。在通道的几何形状和流速的变化可以用于调节液滴尺寸。在这里所描述的微流体设计，与一个直径约为15微米的生成液滴以模仿哺乳动物有丝分裂细胞的几何约束。 <ol><li>光掩模设计和模具制造<ol><li>设计包含三个入口通道31光罩。入口通道1连接到水相，其中包含液滴的内容（见第3节"的重构基本微管紫苑"）。入口通道2连接到油相（参见第2.1节"脂/油PHASE准备"）。使用进口通道3之前的观察（可选）（ 图2A-B稀释乳化液滴额外的脂质/油相）。 注:所有入口通道后跟一个防尘过滤器（2微米频道的迷宫），以捕集粉尘，PDMS粒子和（蛋白质）聚集体，并防止它们阻断微流体通道（ 图2d）。通道75微米宽，并在12.5微米宽结，其中乳液的液滴会形成（ 图2c）的脂质/油相和水相满足。 </li><li>订购并得到薄膜基材上印刷的光掩膜，具有负极性（光掩模将暗带透明设计结构）。 </li></ol></li><li>模具制造<ol><li>制造通过旋涂SU-8 3025光致抗蚀剂模具上使用旋涂机（以500rpm，10秒，随后1800转，45秒）在一个干净的房间环境中的4英寸硅晶片。 </li><li>暴露SU-8涂层通过光掩模的硅晶片。根据制造商的说明来创建40微米厚的〜频道发展晶片。 </li></ol></li><li>制作微流控芯片<ol><li>在使用塑料抹刀舟状容器混合10份的PDMS预聚物与1份固化剂（总〜40克）。含在真空室内约30分钟船状容器的地方的PDMS。以除去气泡。包裹铝箔周围的模具，以形成具有约1cm的直立边缘的杯。 </li><li>倒入PDMS（〜总体积的75％）到模具，留下在真空室内为另一个〜30分钟。 （以除去气泡），并在烘箱中在100℃下固化1小时。 </li><li>旋涂剩下的PDMS到玻片上（在200rpm 5秒接着30秒在4,000rpm下），随后固化在100℃下1小时。使用压缩空气/ N 2 -flow，预清洗是没有必要的载玻片上的灰尘。 注:涂覆的PDMS载玻片可以为麦克风被用于既rofluidic芯片和流动池的生产（参见2.2）。 </li><li>轻轻剥去PDMS关闭使用刀片的模具和冲头的孔（0.5毫米入口，0.75毫米出口）.Corona-处理微流体芯片和使用实验室电晕表面处理机为几秒钟的PDMS涂覆载玻片。 </li><li>放置在微流体芯片上的载玻片（通道朝下）和烤芯片O / N在100℃下（ 图3a）。存储微流控芯片用于在无尘环境几个月。 </li></ol></li></ol> 2.微流控设置注:水包油使用的是微流体设置和上述的微流体芯片产生的乳液液滴。脂质/油相的组合物可以根据实验要求而变化。油增溶脂质将形成在水 - 油界面朝向里面的水的极性头部基团的单层。为了靶蛋白（ 如动力蛋白），以液滴的边界，（生物素基PE）可以加入少量的生物素-磷脂的脂质。这使得生物素化动力蛋白的招募（参见4.1节"动力蛋白靶向滴皮层"），通过链亲和素介导的多聚化。液滴通过加入表面活性剂的稳定化，并存储在PDMS涂覆流动池。 <ol><li>脂质/油相制备方法<ol><li>混合氯仿溶解1,2- dioleoyl- -sn-甘油-3-磷酸-L-丝氨酸（DOPS）和生物素基PE脂质在4:在使用玻璃吸管的玻璃管1的摩尔比（广泛前冲洗，用氯仿吸管使用）。制备总共〜250微克脂类，导致〜0.5毫克/毫升的脂质浓度。 </li><li>仔细擦干用N 2 -flow并随后在真空室内〜1小时的脂质混合物。溶解在矿物油和2.5％表面活性剂的干燥脂质0.5毫克/毫升（使〜500微升）。 </li><li>将在脂质/油样超声浴并超声处理30分钟。在40千赫至完全溶解脂质。 </li></ol></li><li>流动池准备<ol><li>旋涂的PDMS（也见1.3节）装到盖玻璃（以200rpm 5秒接着30秒在4,000rpm下）和载玻片（在100rpm 5秒接着30秒1500转），以沉积均匀层PDMS的。 注:为了获得最佳的成像质量，确保使用盖玻片厚度的显微镜物镜相匹配。在这种情况下:1.5（〜0.17毫米）。 </li><li>固化涂覆的PDMS盖玻璃和玻璃载玻片在100℃的烘箱中1小时。由紧密间隔使流动细胞（〜2mm）的实验室密封膜的薄片（〜3毫米宽）上涂布的PDMS - 玻璃 - 幻灯片。当存储在无尘的环境中，还没有被使用的信道可以在其它时间使用。 </li><li>通过熔化实验室密封膜〜1分钟覆盖流动细胞与PDMS涂覆的盖玻片和密封。在100℃时，按轻轻（ 图3c）。与Valap（ 图3c）关闭实验室密封膜-玻璃-边界。商店流动细胞用于在无尘环境几个月。 </li></ol></li><li> PDMS杯准备<ol><li>对于长期成像，使PDMS杯，通过在PDMS的切片冲切4毫米直径的孔（〜3mm厚） </li><li>电晕处理的PDMS片和PDMS涂覆玻璃盖，并将其放置在彼此的顶部。烤杯PDMS O / N（100°C）（ 图5a）。 </li></ol></li><li>乳化液滴形成<ol><li>监视器上倒光明场显微镜液滴形成。压力控制器连接到使用的PEEK管中的微流体芯片（直径:510微米（外侧）与125微米（内侧））（ 图3a）。填充微流控芯片完全脂质/油相从进口2。 </li><li>推出基于MRB80水相（见第3节"基本的重构微管紫苑4）从入口1控制液滴尺寸通过改变脂质/油相和水相的压力来创建的液滴〜15微米的直径（ 图3b）。 注意:使用该设置中，使用〜800毫巴的脂质/油相和〜200毫巴的水相以产生所需尺寸的液滴。 </li><li>获得所需的微滴大小和所需的量后（每个样品10微升），收集来自出口通道和负载液滴进入流动池（完全填充流动池）。 </li><li>关闭小心使用Valap流动池的端部（不完全封闭流细胞可导致液滴广泛的运动，使得难以图象它们）。另外，防止气泡这导致液滴运动的形成。 </li><li>用异丙醇广泛冲洗的PEEK管使用前和使用后防止样品的交叉污染和堵塞。 </li></ol></li></ol> 3.重建基本翠菊微管注:液滴内容可以根据实验要求而变化。所有缓冲MRB80基于（80毫摩尔PIPES，1mM的EGTA，4mM的MgCl 2的（pH为6.8）），以及所有的样品都在冰上制备。一般情况下，微滴总是包含中心体，微管聚合，除氧剂系统和阻断剂所需的组件（参见3.1节）。微管力发电机和所需的辅助因子和靶向因子如果需要，可以包括（见章节4.1和4.2）。 <ol><li>在乳滴翠菊形成常规设置（ 图3d） <ol><li>在-80℃的准备葡萄糖氧化酶（200毫米DTT和10毫克/毫升的过氧化氢酶20毫克/毫升的葡萄糖氧化酶）混合事先并存储在小分装。 </li><li>制备含有κ酪蛋白，牛血清白蛋白（BSA）一个"阻塞混合"，吐温20和荧光葡聚糖（作为中立标记）（ 见表1）提前，并存储在小aliquoTS在-80℃。防止反复冻融。确保所有组件都新鲜烹制。 </li><li>净化在小分装在-150℃下Moudjou 等 32店作为描述的人类淋巴细胞KE37细胞中心体。 </li><li>在室温下解冻中心体，并在37°C孵育〜20分钟。在使用前要确保正确的微管核。 注:此实验过程中促进微管核。 </li><li>在平均时间，准备"测定混合物"，含有微管蛋白（荧光和"暗"），鸟苷三磷酸（GTP），氧清除剂系统（葡萄糖，葡萄糖氧化酶，过氧化氢酶和1,4-二硫苏糖醇（DTT）），分子力发电机（ 例如微管电动机/交联剂），三磷酸腺苷（ATP）和ATP再生体系（磷酸烯醇丙酮酸（PEP），丙酮酸激酶（PK）和乳酸脱氢酶（LDH））在冰上（ 见表2）。 </li><li>预冷却冰上airfuge转子。降速样品中的冷却airfuge转子（30磅为3分钟）。新增预热中心体（优化中心体的量的目标为含有1-2中心体的液滴）。 </li><li>采用组合结构，以便使用2.3节所述的方法乳化液滴。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 股权集中度 </td><td> 最终浓度（在测定） </td><td> 卷 </td><td> 评论 </td></tr><tr><td>葡聚糖647nm </td><td> 75μM </td><td> 3.75林M（0.6微米） </td><td> 5微升</td><td></td></tr><tr><td> κ酪蛋白</td><td> 20毫克/毫升</td><td> 12.5毫克/毫升（2毫克/毫升） </td><td> 62.5微升</td><td></td></tr><tr><td>吐温-20 </td><td> 5％ </td><td> 0.375％（0.06％） </TD&gt; <td> 7.5微升</td><td></td></tr><tr><td> BSA </td><td> 200毫克/毫升</td><td> 12毫克/毫升（1.9毫克/毫升） </td><td> 6微升</td><td></td></tr><tr><td> MRB80 </td><td></td><td></td><td> 19微升</td><td></td></tr><tr><td></td><td></td><td></td><td> 100微升决赛</td><td>储存在2微升等份</td></tr></tbody></table> 表 1:"阻断混合&#39;的成分阻断混合成分，主轴状结构的水包油乳液液滴的重建所需。为了描述，见正文第3节"基本的重构微管紫苑"。有关材料的详细信息，请参阅" 材料表 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 股票concentratioñ </td><td> 终浓度 </td><td> 卷 </td><td> 评论 </td></tr><tr><td>混合阻塞</td><td></td><td></td><td> 1.6微升</td><td></td></tr><tr><td>微管蛋白</td><td> 500μM </td><td> 30μM </td><td> 0.6微升</td><td></td></tr><tr><td>微管蛋白五百六十一分之四百八十八</td><td> 50μM </td><td> 3μM </td><td> 0.6微升</td><td></td></tr><tr><td> GTP </td><td> 50毫米</td><td> 5毫米</td><td> 1.0微升</td><td></td></tr><tr><td>动力蛋白，TMR </td><td> 178纳米</td><td> 30纳米</td><td> 1.7微升</td><td></td></tr><tr><td>链霉</td><td> 5毫克/毫升</td><td> 200纳米</td><td> 0.4微升</td><td>对于皮质动力蛋白只</td></tr><tr><td>驱动蛋白5 </td><td> 1.6毫米</td><td> 80纳米</td><td> 0.5微升</td><td></td></tr><tr><td> ATP </td><td> 25毫米</td><td> 1毫米</td><td> 0.4微升</td><td>对于仅电机</td></tr><tr><td> PEP </td><td> 0.5M的</td><td> 25.6毫米</td><td> 0.5微升</td><td>对于仅电机</td></tr><tr><td> PK / LDH </td><td> 800 U / 1100ū </td><td> 23 U / 32ū </td><td> 0.3微升</td><td>对于仅电机</td></tr><tr><td> ASE1-GFP </td><td> 400纳米</td><td> 80纳米</td><td> 2.0微升</td><td></td></tr><tr><td>葡萄糖</td><td> 2.5M的</td><td> 50毫米</td><td> 0.3微升</td><td>最后一步</td></tr><tr><td>葡萄糖氧化酶</td><td> 50X </td><td> 1.0倍</td><td> 0.3微升</td><td>最后一步</td></tr><tr><td> MRB80 </td><td></td><td></td><td> X </td><td></td></tr><tr><td></td><td></td><td></td><td> 9微升决赛</td><td></td></tr></tbody></table> 表 2:&#39;测定混合物&#39;的成分。 </strong&gt;测定混合物，主轴状结构的水包油乳液液滴的重建所需的成分。为了描述，见正文第3节"基本的重构微管紫苑"。有关材料的详细信息，请参阅材料表 。 4.引入主轴大会因素注意:在这里所描述的测定中，绿色荧光蛋白（GFP）-tagged S的截短版本用酵母动力蛋白是人为地氨基末端谷胱甘肽S-转移酶（GST）的手段二聚-tag 33。该蛋白质是通过包含该蛋白的两个拷贝的IgG结合结构域的亲和标签进行纯化。此处所使用的变体与四甲基罗丹明（TMR）标记物标记到羧基末端和N-末端的SNAP-标记用于生物素化的纯化的蛋白质。对于结构的详细信息，请参见Roth 等人31。净化详情，请参阅录制K-Peterson 等。33。 GFP-生物素动力蛋白-TMR可以通过链接动力蛋白对生物素基PE脂质（ 图3e）生物素-链霉抗复合物的形成来定位到液滴皮质。 <ol><li>动力蛋白靶向液滴的Cortex <ol><li>包括绿色荧光蛋白-生物素-动力蛋白-TMR到"测定混合物&#39;（ 见表2）为30nM的终液滴浓度。包括链入"测定混合物&#39;（ 见表2），在200纳米的最终液滴浓度。 注:动力蛋白依赖于ATP水解为微管逐步运动。因此，包括ATP和一个ATP再生系统（含有PEP和PK / LDH）到"测定混合物&#39;（ 见表2）。 注:重组全长S.酵母 GFP-Cut7（驱动蛋白-5）好心的迭实验室（中心分子生物工程，德累斯顿工业大学，德国德累斯顿）提供的，见。重组全-Length组氨酸标记S.裂殖酵母的GFP-ASE1惠赠从松斯实验室（瓦格宁根大学，瓦格宁根，荷兰）提供，并添加到"测定混合"在浓度为80纳米。 </li></ol></li></ol> 5.成像注:在实验过程中，微管生长可通过改变温度进行控制。在选择的摄像条件下，权衡有高质量的成像和监测长时间时段主轴组件的能力之间进行。比较不同条件下的主轴形成的动力学，具有不同含量的液滴可以混合，并在同一流道成像。 <ol><li>基本图像设置<ol><li>图像中的温度控制的环境中使用一个封闭的腔室。可视化后〜30分钟，在26℃个别微管。而成像，通过提高温度高达约30℃促进微管生长。 注:在S下面ettings特定于我们的显微镜装置，可与不同的系统和不同的操作软件而异。此处所述的所有试验都成像与旋转盘共聚焦头倒立机动系统上，并使用图像处理软件，如安道尔的iQ 3.1操作。获得了100倍的油浸物镜和EMCCD相机的所有图像。 </li><li>设置激励激光器488，561和641纳米至大约〜10％的强度。进入"收购"面板，点击"AOTF"选项卡和幻灯片激光强度为10％。点击"记录"保存设置。 </li><li>对于z -projections，采取与1微米的间隔（约20张/液滴）堆栈。进入主"摄像头"面板中，单击"编辑Z&#39;，并设置&#39;Z一步"为1.0，点击"下一步"保存设置。 </li><li>通过点击"相机"选项卡上的"收购"面板中设置最大线性EM增益和EM增益条滑动到300设置曝光次，以在同一个标​​签200毫秒。点击"记录"保存设置。 </li></ol></li><li>实时成像<ol><li>对于实时成像，使用软件控制使ž-projections每2分钟。 1-2小时的通过减少曝光时间来〜100毫秒的持续时间。 （如在5.1.4中描述），并增加Ž-intervals至2μm（如在5.1.3节）。设置时间序列中主要的"摄像头"面板中，单击"重复T&#39;，并设置为2分钟。间隔为120分钟的总时间。 </li><li>对于实时测定，使用PDMS杯代替常规流动池，以确保液滴尽可能不动。 </li></ol></li><li> 2条件联合成像<ol><li>产生使用微流体液滴和储存在冰上将PDMS涂覆载玻片的顶部。这可以防止微管成核，直到第二组液滴已经形成。产生第二组液滴之前，冲洗的PEEK管中，用微流控芯片MRB80并完全重新填满用脂质/油相的微流体芯片。 </li><li>生成具有和不以便用不同颜色的液滴区分荧光葡聚糖（或使用葡聚糖具有不同波长的荧光团）的液滴。生产第二批液滴后，轻轻吹打混合液滴和加载到一个单一的流动池/ PDMS杯。 </li></ol></li><li>图像分析。 <ol><li>通过分析斐济（开源软件可在线）34图像。 </li><li> "hyperstacks&#39; - - &#39;栈hyperstack"现场试验，使用"图像"转换成堆栈hyperstacks。设置的z切片和时限的正确数目。 </li><li>为了使Z-预测，选择"图像" - "栈" - "Z-工程"。选择"最大强度"的预测。 </li><li>对于三维重建，首先要到"图像" - "属性"，并设置与像素heigth，像素深度和FR的适当的像素AME区间。点击"确定"。选择"图像" - "栈" - "三维工程"，选中"插值"复选框，然后单击"确定"。 </li></ol></li></ol>

<ol>
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The authors declare that they have no competing financial interests.

描述的方法最近在这里本努力以重构基本有丝分裂纺锤体中的水包油乳液液滴。几何范围内研究纺锤体组装提供了许多优势。重要的反馈机制的有丝分裂纺锤体的微管，并在它们被组装的几何约束之间存在。微管可以产生时，他们生长成几何边界，这可导致有丝分裂纺锤体位移推力。此外，成长为一个物理屏障可诱发微管灾难。另外，在密闭几何形状内主轴组件可导致单个组件，例如微管蛋白，这又直接影响微管生长速率36的逐渐枯竭。这些是心轴组件的所有必要决定因素，其可以使用此处所描述的测定法进行研究。 所描述的试验是小各色浓度非常敏感液滴组成部分NCES。这是为微管蛋白，其中微小的浓度差异可导致在任一过慢或过快微管生长的情况。在这种情况下，微管生长速率往往仍然可以通过在实验的第一〜30分钟期间调节温度校正。有丝分裂纺锤体组装和定位也对在（皮层）力产生的浓度变化非常敏感。这很可能是依赖于纯化的组分的浓度，纯度和活性，并且需要为每个新纯化的蛋白质进行仔细滴定。 此外，某些权衡有在成像设置进行，根据测定的性质。最初，高含量的水溶性荧光微管蛋白二聚体使得难以图像个别微管。作为微管变长并可溶性微管蛋白缓慢地耗尽，信噪比逐渐变高，并允许individu的摄像微管人。在对比开放系统设置，几何约束可防止荧光分子和氧清除剂系统组件的堆积贮存器内的交换，导致显著漂白。尤其用于监视主轴组件和定位在一段时间，需要具有低光强度的图像，即使是在一个有效的除氧剂体系的存在。 这些方法使体外简化纺锤状结构的自下而上重建。除了 ​​这里介绍的组件，已描述了许多其他因素影响双极纺锤体的形成，定位，定向，整形等 3。该系统可以进一步扩展研究的附加力产生的个体和组合的效果。重要贡献者纺锤体的形成是目前缺席这个系统是有丝分裂染色体。有丝分裂的染色质已被证明由（1）chromokinesins直接纺锤体形成，可以产生所谓的"极性喷出力&#39;3，（2）一个RanGTP梯度由染色质结合RanGEF RCC1 38的形成，（3）着丝粒，可与（解聚交互）微管39和（4）的染色质本身，它可以相对微管40之间在作为机械弹簧作用。 最后，上述系统目前提供了活动和有限的时间和空间控制引入力发电机的国产化。在细胞中，许多主轴组件因子定位于特定隔室或限制时间窗口内是活动的。一个突出的例子是动力蛋白的活性，这是专门在C的后侧丰富线虫单细胞阶段的胚胎，以促进非对称主轴定位和细胞分裂。在这个系统中，我们目前正在探索模仿这种T的可能性emporal和非对称使用活性的方法从光遗传学技术借来的。此外，由于是为C的情况下线虫胚胎和用刚性细胞壁的细胞，许多细胞不允许有丝分裂围捕。几何约束的形状会对作用在主轴41上的力产生根本性的影响。因此这将是重要的，以重建在非球形液滴主轴组件和定位，以及，可能使用了允许成形和乳液的贮存液滴42,43更复杂的微流体系统。最后，在组织中，细胞 - 细胞和细胞 - 基底信令和附件提供可以被翻译成指向主轴定向外部线索，如上皮细胞经常观察到细胞和干细胞。所有这些专业方面都没有考虑到目前的研究，并可能提供未来的挑战，建立对有丝分裂纺锤体assemb更体内般reconstitutionsLY和定位。

在有丝分裂，复制的基因组的染色体在细胞赤道组织，以确保在新形成子细胞姐妹染色单体的相等分布。染色体定位和隔离是通过自己的依恋介导的从两个对立的起源中心体纺锤体微管。除了 ​​确保忠实染色体分配，有丝分裂纺锤体的取向也决定了细胞分裂平面1。细胞分裂的协调的方向是在发展的许多阶段和组织稳态2至关重要。具体地，调节有丝分裂纺锤体定位可导致细胞内容物的非对称分布，甚至产生不同尺寸2的子细胞。有丝分裂纺锤体组装和取向开始在前期和由合作并拮抗力发电机3,4之间复杂的相互作用编排。生成的部队组成的BOTH推力和拉力。这些力都从与小区皮质主轴接触以及从主轴内发起，并且可以通过微管动力学以及由分子马达和交联剂（ 图1）来生成。 推力可以在微管生长对刚性结构如细胞皮层产生。取决于系统内产生的总阻力，这可能会导致有丝分裂纺锤体（低力）的重新定位或触发微管屈曲或灾难（高力）5-8。可以通过按压来产生的力的量取决于微管长度，由于长度的微管屈曲9的重要决定因素。此外，从一个中心体发起的微管可推压在其他中心体，已被建议的用于驱动在早前期3,10中心体分离的机构。 在广告DITION力推通过种植微管产生的力，微管两端接触细胞皮层也可以调解拉力11。已经从多个实验室研究表明，皮质力发电机的控制活动所需的有丝分裂纺锤体的体内 1的非对称定位。必要对这些拉力是皮质本地化胞质动力蛋白（以下简称为"动力蛋白&#39;），一个负端定向微管马达蛋白8,12,13。例如，在芽殖酵母，皮质动力蛋白沿着皮质14滑动微管晶格结果在马达依赖微管的侧向相互作用。但是，也可以通过动力蛋白，形成最终的附件，微管解聚8的能力而产生的皮质拉力。通过微管收缩产生的能量可导致力，其通常是数量级高一个数量级（约50 PN（参考15 </s达&gt;））不是由单个马达蛋白产生的力（〜7-8 PN（参见16））。以微管解聚皮质动力蛋白的最终依恋促进主轴定位在芽殖酵母17和C.线虫 13。无论是电机相关的滑动和微管解聚驱动两种皮质拉力可以合作或在体内相互排斥是目前尚不清楚。 除了微推力和动力蛋白介导的皮质拉力，中心体定位在有丝分裂纺锤体内许多其他的蛋白质控制。驱动蛋白-5-马达，例如，存在作为可交联反平行极间微管，导致向外滑动力18-20的产生四聚体。驱动蛋白-5马达家族的成员都需要在研究的所有真核生物中心体分离和双极主轴组件，与C的异常线虫<html> （在参考21中综述）。 此外，同样是公知的ASE1 / PRC1家族的扩散交联剂本地化在体内 和体外 22-27重叠极间微管的微管的区域。由重叠微管区域的ASE1驱动膨胀所产生的力大到足以抵消驱动蛋白14介导的滑动力体外 28,29。在细胞中，需要在主轴midzone 25-27,30重叠的微管的区域，这表明通过扩散交联剂产生的力可以显著向主轴组织的稳定形成ASE1 / PRC1。最后，从有丝分裂染色力和动粒通过各种途径3影响有丝分裂纺锤体组装和定位。因为这些组件不是这里所描述的重建试验的一部分，它们将不进行详细的讨论。 jove_content"&gt;在活细胞对上述不同的分子组件和力量是如何在纺锤体组装和定位合作存在不同的理论，但我们仍远远没有一个定量的了解。此外实验，体外实验用纯化的组件提供了一个功能强大的路线，以帮助实现这一目标。在这里，我们提出一个视觉引导的最近发表的方法的调整和扩展版本（Roth 等人 31），其中基本的主轴在水-油乳化液滴重组，从一个最少数量的部件，利用微流控技术，与那些可比有丝分裂细胞的大小产生的球面的液滴。这些液滴内，纯化的中心体和微管蛋白可以为了研究微管翠菊动力学，力产生和翠菊-翠菊相互作用来结合，通过引入皮层（如动力蛋白）和极间（如驱动蛋白-5 / ASE1）力发电机，我们重构一个开始类似于体内情况日益复杂的纺锤状的结构。

<table><tbody><tr><td>&lt;strong&gt;1. Preparation of microfluidic chips&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Photomask (Photomask on film substrate)</td><td>Selba S.A. Switzerland</td><td></td><td></td></tr><tr><td>SU-8 3025</td><td>MicroChem</td><td></td><td></td></tr><tr><td>Silicon wafer (4 inch silicon wafer p/Boron &amp;lt;1-0-0&gt;10-20 &amp;Omega;-cm, 500-550 &amp;mu;m, SSP, with 2 flats)</td><td>WRS Materials</td><td>4P0SSP-005</td><td></td></tr><tr><td>Spin coater&amp;nbsp;</td><td>Polos</td><td>SPIN150I</td><td></td></tr><tr><td>PDMS pre-polymer RVT615 A+B</td><td>Lubribond</td><td>9481</td><td></td></tr><tr><td>Corona treater</td><td>Electro-technic products</td><td>BD-20ACV</td><td></td></tr><tr><td>&lt;strong&gt;2. Microfluidic setup&lt;/strong&gt;</td><td></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>DOPS (1,2-Dioleoyl-sn-glycero-3-phospho-L-serine)</td><td>Avanti Polar Lipids</td><td>840035</td><td></td></tr><tr><td>Biotinyl PE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl))</td><td>Avanti Polar Lipids</td><td>870285</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>650498</td><td></td></tr><tr><td>Glass pipets</td><td></td><td></td><td></td></tr><tr><td>Glass tubes</td><td></td><td></td><td></td></tr><tr><td>Vacuum pump</td><td>Laboport</td><td>KNF</td><td></td></tr><tr><td>Vacuum chamber</td><td>Kartell</td><td>Polypropylene Vacuum Desiccator</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M5904</td><td></td></tr><tr><td>Surfactant (Span80)</td><td>Sigma-Aldrich</td><td>85548</td><td></td></tr><tr><td>Sonicator</td><td>Branson</td><td>M2800H</td><td></td></tr><tr><td>Coverslips</td><td></td><td></td><td>24 mm x 60 mm, thickness 1.5</td></tr><tr><td>Glass-slides</td><td></td><td></td><td>26 mm x 76 mm</td></tr><tr><td>Puncher</td><td>Harris Uni-Core</td><td>135840/135841/135843</td><td></td></tr><tr><td>Laboratory sealing film</td><td>Parafilm</td><td></td><td></td></tr><tr><td>Valap (Vaseline, lanolin, paraffin wax melted at equal concentrations)</td><td></td><td></td><td>Home made</td></tr><tr><td>Brightfield Microscope</td><td>Leica</td><td>PMIRB&amp;nbsp;</td><td>Any inverted brightfield would suffice</td></tr><tr><td>Pressure controler</td><td>Fluigent</td><td>MFCS-FLEX-4C-1000</td><td></td></tr><tr><td>PEEK Tubing</td><td>Cluzeau Info. Labo, France</td><td></td><td></td></tr><tr><td>Microfluidics vials (Tubes Micrew 0.5 ml and 1 ml)</td><td>VWR, The Netherlands</td><td></td><td></td></tr><tr><td>&lt;strong&gt;3. Reconstituting spindle formation and positioning&lt;/strong&gt;</td><td></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>Dextran-647 nm (Dextran, Alexa Fluor 647, 10,000 MW, Anionic, Fixable)</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>BSA - Bovine serum albumin</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Glucose (D-(+)-Glucose)</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Glucose-oxidase (Glucose oxidase from Aspergillus niger)</td><td>Sigma-Aldrich</td><td>G6125</td><td></td></tr><tr><td>DTT (DL-dithioltheitol)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr><tr><td>Catalase (Catalase form bovine liver)</td><td>Sigma-Aldrich</td><td>C9322</td><td></td></tr><tr><td>Tubulin (Tubulin from bovine brain)</td><td>Cytoskeleton Inc.</td><td></td><td></td></tr><tr><td>Tubulin-488/561 (HiLyte-488/Rhodamine-labeled tubulin from porcine brain)</td><td>Cytoskeleton Inc.</td><td></td><td></td></tr><tr><td>GTP (Guanosine-&#039;5-triphosphate, sodium salt hydrate)</td><td>Sigma-Aldrich</td><td>51120</td><td></td></tr><tr><td>&amp;kappa;-casein (&amp;kappa;-casein from bovine serum milk)</td><td>Sigma-Aldrich</td><td>C0406</td><td></td></tr><tr><td>Airfuge (Air-driven ultracentrifuge)</td><td>Beckman-Coulter</td><td>CLS</td><td></td></tr><tr><td>&lt;strong&gt;4. Introducing spindle-assembly factors&lt;/strong&gt;</td><td></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>Neutravidin</td><td>Sigma-Aldrich</td><td>A2666</td><td></td></tr><tr><td>ATP (Adenosine-&#039;5-triphosphate, disodium salt hydrate)</td><td>Sigma-Aldrich</td><td>A9187</td><td></td></tr><tr><td>PEP (Phospho(enol)pyruvic acid monosodium salt hydrate &amp;ge;97% (enzymatic))</td><td>Sigma-Aldrich</td><td>P0564</td><td></td></tr><tr><td>PK/LDH -(Pyruvate kinase/lactic dehydrogenase enzymes from rabbit muscle)</td><td>Sigma-Aldrich</td><td>P0294</td><td></td></tr></tbody></table>

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.

在前面的章节中给出的方法允许纺锤状结构的使用水包油乳液液滴的几何限制日益复杂的重建。本节描述代表结果定性证明这些测定的能力。 在有丝分裂中，当双极纺锤体组装，细胞向上舍形成球体的直径为大约15微米，作为人类细胞中测量。这个特性有丝分裂细胞的形状提供了一个几何边界，这两个限制并引导锭子大小和方向35,36。微流体技术，提供几何约束的水平准确类似于在活细胞中观察到的情况，因此，允许在体外有丝分裂纺锤体的自下而上重建。 图4a，左面板）内扩散。约20-30分钟后，将第一微管变得微管生长针对在所有方向上皮层可见和中心体扩散变得受到限制。与中间长度（约50液滴直径的比例）的微管紫苑能（立体）互相排斥另一种，与微管推着对方，其他的中心体和皮质。这导致在一个典型的"双极"纺锤状与两个中心体彼此相对（ 图4a，中间面板）布置。当微管生长比液滴直径的约50％长计，该中心体得到进一步推到液滴的相对边界，与微管 ​​沿液滴皮层（ 图4a，右图）生长。要注意，在这些测定微管生长速率是温度和微管蛋白的浓度非常敏感是重要的。因此，这些参数将强烈影响时，首先观察核，当稳态翠菊位置到达的时间。 在细胞中，皮质拉力通过微管加末端和Cortex-相关的动力蛋白之间的承重附件的形成产生。在动物细胞中，此关联取决于Gαi/ LGN /的NuMA复杂，这是通过Gαi1的N端豆蔻酰化定位到质膜。在这些重组测定中，Gαi/ LGN /的NuMA复杂的要求是由通过直接耦合动力蛋白对生物素化的脂质旁路形成生物素链霉亲和素复合物。这些键是相对稳定的（K D〜10 -14 M），37和快速形成（通常在后乳剂液滴形成10分钟，在此之前的微管成核变得明显）（ 图4b）。皮质动力蛋白的存在下，中心体通常保留一个更中心的位置，而在没有动力蛋白的，中心体推到液滴皮层（ 图4c）的相对两侧。我们有理由这是两方面的影响，其中预防和制止微推力的结果。 （1）动力蛋白直接促进微管灾难，从而限制微管长度，并防止过 ​​度的微管屈曲，和（2）大脑皮质的拉力导致净定心力量于个人紫苑8，其抵消翠菊-翠菊斥力。 扩散性交联剂ASE1诱导˚F趋向orces增加反平行的微管29的重叠区域。与此相一致，在ASE1的存在（和不存在动力蛋白的）中心体被紧密连同ASE1定位到捆绑极间微管（ 图4d）中找到。动蛋白-5系列的成员由主轴内提供的推力推动中心体分离（见介绍）。在Cut7（ 酿酒酵母驱动蛋白5直向同源物）的存在，中心体推到乳液液滴的相对侧，即使在ASE1（ 图4e）的存在。通过组合皮层和极性间力发电机，复杂程度，可以进一步在这些实验中增加，最终导致双极有丝分裂纺锤体组装的全面理解。这些结果的详细的定量描述将在未来可用的（Roth等人，在制作手稿）。 <p class="jove_content" fo:kee对 - together.within页="1"&gt;除了观察中心体的位置以固定的时刻在时间上，以及与他们的行为的微管的观测的长度，可以通过在下面的定位方法获得的有价值的信息时间。通过减少曝光时间和激光强度，现在有可能跟随翠菊定位在2分钟的时间间隔为至少1小时，只有约30％的漂白。这允许通过集中从自由扩散中心体翠菊装配和定位的监控（0分钟）（12分钟），以分散的紫苑（36分钟，并进一步）（ 图5c）。这有利于双方稳态行为和纺锤体组装动力学的研究。用相同的样品中结合具有不同内容的液滴，它是，例如，可以直接在微滴具有和不具有皮质力产生（ 图5b）比较中心体定位和主轴组件动力学。 图1: 强制作用于有丝分裂纺锤体的若干力产生分子作用于有丝分裂纺锤体促进纺锤体形成和定位的微管。该生长到细胞皮质微管生成在中心体的推力。皮质动力蛋白（紫色）捕获解聚微管，并产生在中心体的拉力。在主轴，驱动蛋白-5 / Cut7马达蛋白提供反并联微管向外滑动力，而PRC1 / ASE1家庭的交联剂产生相反的向外滑动的力量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54278/54278fig1large.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/54278/54278fig2.jpg" /> 图2:含有4微流体芯片4英寸光罩的微流控芯片设计 （A）的设计。 （B）单芯片的详细表示。芯片包含一个出口通道和三个入口通道后跟一个防尘过滤器。入口通道1包含水相，信道2中的脂质/油相和信道3可以用来稀释用另外的脂质/油相所形成的小滴。 （C），其中的脂质/油相（从顶部和底部来）与水相（从左侧来）满足结的详细表示。交界处，液滴就会形成并且朝右侧的出口通道流动。 （d）与2微米频道的粉尘过滤器的详细表示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54278/54278fig2large.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/54278/54278fig3.jpg" /> 图 3: 方法论水油包水乳液液滴的形成 （A）微流控芯片和微流体管道上一个明亮的视野显微镜。同时拒水和脂质/油相管分别连接到芯片的入口1和2。在微流体芯片，其中水和脂质/油相适应（B）限制。通过改变压力，液滴尺寸可以得到控制。 PDMS涂覆流动细胞（C）的设计。装载液滴进入流动池后，将开口端封闭额外Valap。 （D）的液滴的形成原理。液滴用于封装中心体，微管蛋白和有丝分裂纺锤体的形成需要另外的组分。皮质-动力蛋白靶向的（E）示意图。生物素化（生物）动力蛋白是通过链亲和素（STRP）针对生物素化脂质。 <aHREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54278/54278fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54278/54278fig4.jpg" /> 图 4: 日益增长的复杂性主轴，Reconstitutions的代表性结果 （A）包含两个中心体显示出短期，中期和长期的微液滴实例的最大强度的预测。中心体和微管通过加入10％HiLyte-488标记的微管蛋白的可视化。比例尺= 10微米。在没有GFP-生物素动力蛋白-TMR（ 二 ）本地化（ -左）和链霉（STRP）的存在（+，右）。皮质GFP-生物素动力蛋白-TMR的或存在（+右） - （C）的微管翠菊定位在不存在（左）。 （D）微管紫菀（左图，GREEN在ASE1的存在下）定位（中间面板，红色）。在ASE1和Cut7（驱动蛋白-5）（中间面板，红色）的存在（E）微管紫菀（左图，绿色）定位。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54278/54278fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54278/54278fig5.jpg" /> 图5:成像紫苑动态定位 在体外 （A）一PDMS杯，让液滴成像长达数小时的设计。 （B）的生成，存储和液滴成像（传输）包含不同的内容，通过荧光葡聚糖的情况下（左）包含（右）（Alexa的647）所示。 （℃）60分钟的时间推移的单平面图像（2分钟的间隔）从广告的Z栈（2微米的距离）被包含一个中心体roplet。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54278/54278fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets at JoVE.com

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

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 ...

reconstitution-basic-mitotic-spindles-spherical-emulsion

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9.7K 次观看.  Delft University of Technology. 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.

视频: Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

科研

生物化学

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

化学

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

用完全纯化的组分重构Msp1提取活性

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

微管触觉的自组装

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

生物工程

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

颗粒模板化乳化可实现无微流体液滴检测

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the ...

生物学

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

细胞骨架对小鼠卵母细胞核形状和凝聚物动力学的影响

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

作者聚焦：阐明小鼠卵母细胞中机械转导和核搅动的动力学

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

跨空间尺度捕获基于细胞骨架的小鼠卵母细胞核搅动

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

基本的有丝分裂纺锤体的重组在球形乳液液滴

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此视频中的章节

巨囊泡封装微球通过一个水包油乳液的离心制备

无细胞爪卵萃取物微乳液细胞周期振荡的重建

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

用完全纯化的组分重构Msp1提取活性

微管触觉的自组装

在膜上重构隔膜组件以研究生物物理性质和功能

体外重组巨型单层囊泡内的肌动蛋白细胞骨架

颗粒模板化乳化可实现无微流体液滴检测

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

跨空间尺度捕获基于细胞骨架的小鼠卵母细胞核搅动

基本的有丝分裂纺锤体的重组在球形乳液液滴

摘要

探索更多视频

此视频中的章节

巨囊泡封装微球通过一个水包油乳液的离心制备

无细胞爪卵萃取物微乳液细胞周期振荡的重建

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

用完全纯化的组分重构Msp1提取活性

微管触觉的自组装

在膜上重构隔膜组件以研究生物物理性质和功能

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

颗粒模板化乳化可实现无微流体液滴检测

在支持的脂质双层上重建膜系的最小肌动蛋白皮质

跨空间尺度捕获基于细胞骨架的小鼠卵母细胞核搅动

体外重组巨型单层囊泡内的肌动蛋白细胞骨架