Name: characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis
Uploaded: 2014-10-30T13:00:00.000+00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

We thank Ge Yang, Gaudenz Danuser, Khuloud Jaqaman, and Daniel Whisler for assistance with the adaptation and development of the software to quantitate cargo mapping analyses, and Emily Niederst for help in making the microfluidic devices. This work was supported in part by NIH-NIA grant AG032180 to L.S.B.G., and the Howard Hughes Medical Institute. L.S. was supported in part by a NIH Bioinformatics Training Grant T32 GM008806, S.E.E. was supported by a Damon Runyon Cancer Research Foundation Fellowship, NIH Neuroplasticity Training Grant AG000216, and by a grant from The Ellison Medical Foundation New Scholar in Aging Award, G.E.C was supported by an NIH/NCATS&#160;1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.

所有实验均进行以下批准的协议，并根据对人道护理研究动物的机构准则。新生儿小鼠断头处死。 1.制备微流体装置的用于细胞培养<ol><li>所描述的哈里斯和他的同事为17-19海马神经元的生长，准备聚二甲基硅氧烷（PDMS）微流控设备。下面是分别适合于货物映射协议的一些修改。 注意:微流体装置也可商购（材料清单），从而获得了制造设施是没有必要的。 </li><li>通过洗涤他们三次，用丙酮，然后洗涤三次用100％乙醇和水洗涤三次准备号1.5盖玻片（24×40毫米）。商店盖玻片在水中，在4℃直至使用。这些治疗有助于确保盖玻片清洁碎片可能与CEL的镀干扰的ls，污染，和存活。 注:丙酮和乙醇摄入的易燃和危险情况下的皮肤接触（刺激物），眼睛接触（刺激物），吸入。根据官方的规章处理。 </li><li>当准备使用时，将一只玻璃盖在每个微流体装置60毫米的细胞培养皿中，并让其风干揭露为45分钟的生物安全柜中，以避免污染。 </li><li>后设备从主人切出冲头已经被切以创建储层（ 图1A，B）所示，将它们与微流体通道侧式生物安全柜装有UV灯45分钟进行灭菌 ​​的内部。 注:将时间超过2小时，在UV处理设备可能会影响PDMS中的完整性。 </li><li>通过放置到每个盖玻璃60毫米的塑料细胞培养盘内的一个设备组装装置，使得微流体通道朝下。轻轻推压至顶Of显示装置（避免接触微通道），以提供该装置的非永久密封到玻璃盖。涵盖各60 MM细胞培养皿的盖子。 </li><li>用微量中，加入细胞培养级水中，顶部的两个微流体储层（ 图1A中的1和2），从而允许水流过水合设备。用微量中除去水并加入1mg / ml的聚-L-赖氨酸进入微流体储存器1（200微升）和2（100微升）。音量差是允许的聚L-赖氨酸流过微通道（ 图1A，B）。 </li><li>为确保内部60毫米细胞培养皿的设备不翻倒内37℃的孵化器，将装有这些设备360毫米细胞培养皿至150毫米的塑料细胞培养皿中，并盖上盖子的菜。孵育O / N在37℃培养箱中5％的CO 2。通常在90％以上的信道是每个器件可用做不包含任何气泡或物理堵塞。 </li><li>第二天，更换聚-L-赖氨酸与水和离开设备在培养箱中培养至少1小时。重复此洗涤三次。除去水和添加的Neurobasal-A培养基（含2％的B-27补充剂和500μM的含有GlutaMAX）到每个设备。离开O / N在37℃培养箱中培养5％的CO 2。 </li><li>第二天，如下所述板状海马细胞。 </li></ol> 2.电镀小鼠海马神经元主要在微流体装置注:培养海马神经元的新生大鼠制备先前已发表 ​​在朱庇特25。下面是分别适用于平板小鼠海马神经元微流体装置的一些修改，这是最佳的转染。 <ol><li>前小鼠脑解剖，预暖的Dulbecco改良的Eagle培养基（DMEM），含有10％胎牛血清胎牛血清（FBS;不含抗生素），混合物A（125毫克DL半胱氨酸，125mg的牛血清白蛋白（BSA）和3.125克D-葡萄糖在500ml磷酸盐缓冲盐水（PBS）中;过滤除菌通过0.22微米的过滤器）和脱氧核糖核酸酶I溶液（DNA酶I:50毫克，DNA酶I和0.5的100ml Hank氏平衡盐溶液（HBSS）中过滤1.2M的硫酸镁溶液消毒通过0.22微米的过滤器），在37℃的水浴中。 <ol><li>预暖的Neurobasal-A内的37℃培养箱中，用5％CO 2的生长培养基中平衡的pH值。 </li></ol></li><li>根据既定方案26 1-3日龄乳小鼠海马解剖。保持海马在15ml锥形管中含有10ml夹层缓冲液（HBSS中含有10mM HEPES pH为7.4，50mM葡萄糖，100U / ml青霉素和100μg/ ml链霉素），在冰上。把高达的15毫升锥形管4海马。 </li><li>执行剩余的协议是在无菌条件下s的生物安全柜内。 </li><li>稀释在5ml的混合物A，涡流的45个单位的木瓜蛋白酶，并孵育5分钟，在37℃，直至木瓜蛋白酶粉末溶解。加入1 ml的DNA酶I溶液。过滤通过0.22微米的注射器过滤器单元的组合。 </li><li>小心吸HBSS"15毫升锥形管含海马。加入10毫升冷的（4℃）的HBSS到海马，并让他们沉降到管底部。小心15毫升锥形管吸HBSS。 </li><li>加入1 ml木瓜蛋白酶/ DNA酶I混合至多达4海马孵育20-30分钟，在37℃。倾斜的锥形管，每隔5分钟去洗澡海马与组合。可替换地放置海马在37℃水浴摇床上轻轻振荡（〜100rpm下）。 </li><li>抽吸木瓜蛋白酶/ DNA酶I混合并用含有10％FBS的预温热的DMEM（37℃）洗涤海马两次。 </li><li>第二次洗涤后，加2 ml预热的DMEM中含有10％FBS至海马和手动磨碎海马通过移起来，并使用1毫升移液管尖，以解离的神经元向下〜10-12倍。 </li><li>让磨碎沉降〜1分钟，因为这将允许非解离神经细胞沉降到锥形管的底部。传送含有解离的神经元的上清液至新的15毫升锥形管中，避免较大的组织已经固定在该管的底部的传递。 </li><li>自旋细胞在1000 XG 2分钟，小心地取出上清液，而不会干扰细胞沉淀。轻轻吹打重悬在80微升的Neurobasal-A培养基中的细胞。 </li><li>从微流体储存器1和1&#39;中删除介质。适用于20微升细胞（〜275000），以微流体水库1座，并让他们流过。放置设备在37℃培养箱中，用5％CO 2的20分钟。 </li><li>看显微镜下，以确保细胞粘附在玻璃盖。添加的Neurobasal-A培养基来顶掉所有的水库。返回装置与细胞在37℃培养箱中，用5％的CO <sub&gt; 2。 </li><li>检查设备的每个〜2-3天顶过水库的Neurobasal-A含有2％B-27的补充（不含有GlutaMAX），以避免蒸发。神经细胞开始通过微流体通道电镀后的轴突伸长〜2-3天。受试者神经元接种后转染通常7-10天。 </li></ol> 3.转染海马神经元生长的微流控设备<ol><li>每台设备，准备1.2μL脂质体2000年30微升的Neurobasal-A混合，和0.5微克荧光货物融合基因在30微升的Neurobasal-A混合，并培育5分钟在室温。添加脂质体的混合的DNA混合，并孵育20分钟，在室温。加入60微升含4％B-27补充DNA的Neurobasal-A的/脂质体混合，并通过弹管混匀。 </li><li>从微流体水库2和2&#39;删除所有媒体和认真填写了含有2％B-27的补充井的Neurobasal-A不洒在中INTØ其他舱室。 </li><li>从微流体水库1和1&#39;取出介质。加入120微升的DNA /脂质体混合到微水库1座，并让它流。返回装置至37℃培养箱，用5％CO 2的3-4小时。 </li><li>从微流体水库1和1&#39;删除所有媒体和用Neurobasal-A含有2％B-27的补充替代24小时（或直到准备映像）。典型地，5-7个轴突，通过微通道长的那些条件，这代表约1-3％的转染效率与已发表 ​​的对海马培养细胞26的效率一致的下转染。 </li></ol> 4."货物映射"分析<ol><li>货物运输的实时成像<ol><li>在倒置落射荧光显微镜的光配备了37℃培养箱中培养和CO 2控制室进行拍摄。 </li><li>安装在一个MICR成长的海马神经元的盖玻片ofluidic设备上的显微镜载物台。为了避免神经元死亡，工作迅速，保持在显微镜的样品，不超过1小时。 </li><li>使用高分辨率的物镜（100X），发现转染的细胞，通过微流体腔室的通道延伸，并记录在其中转染的轴突中发现的信道的数量的轴突。算使用手动式计数器的信道数。 <ol><li>对于活运动和随后的共定位分析的最佳记录，选择成长相对平坦的通过渠道，可以重点进行最囊泡的成像轴突。 </li></ol></li><li>水塘2和2&#39;与1毫升塑料移液管除去大部分培养基。为了便于固定图像与移动轨蹄的上kymographs随后的对齐，对齐的视场中的微通道的右侧边缘成像（ 图1B中的C）中。 </li><li>为了方便菲克桑G拍摄过程中的样品，进行实时成像两个人。另外，如果实时成像是由一个人进行，使用配有漂移校正的显微镜系统。 </li><li>开始摄像活货物异动，具体到货物的被分析的传输动力学时间推移规格（显微镜的规格和设置用于成像的YFP-朊病毒下在图2和材料清单的说明中指定）。 </li><li>经过足够活运动数据已经收集，有一个人填满水库2和2&#39;与在PBS中预温4％多聚甲醛含有0.04克/毫升蔗糖以固定细胞。避免接触微流体装置，因为这样会破坏实时成像的焦点。有而固定剂被加入的其他人调整焦点。固定是成功的，当移动立即停止观察。添加固定剂对储层1和1&#39;之后。 注:临时photoblea货物荧光清也可能被观察到，但荧光仍然存在后，如果染色马达蛋白完成（下一步）。 注意:多聚甲醛吸入有害，皮肤接触和吞食。刺激眼睛，呼吸系统和皮肤。根据官方的规章处理。 </li></ol></li><li> IF染色马达蛋白的共定位分析 注:对于马达蛋白的相对水平（如通过抗体染色测定），相关联的单独移动的货物的定量，验证和滴定的抗体，以确保所述的抗体 - 抗原结合是饱和的。这是通过确定使用神经细胞，其中感兴趣的蛋白质已被任敲除或敲低对抗体的特异性进行，并且通过执行IF分析随着抗体浓度。为阴性对照，神经元染色，在不存在第一抗体的第二抗体。更详细抗体的验证的说明可在Encalada 等人 16和Szpankowski 等 20中找到。 <ol><li>删除从显微镜载物台在微流控设备增长的海马神经元的盖玻片。不从盖玻片脱离微流体装置中，所述微通道的需要，以叠加的火线和固定的图像保持完整。执行在湿度室中的下列步骤。 </li><li>以确保溶液的流入微通道，至少30微升保持油藏1,1之间的缓冲体积差&#39;和2,2&#39;。在所有后续步骤。例如，添加100μl的培养基体积对微流体贮存器2，2&#39;，和70微升培养基体积对微流体储存器1,1&#39;。 </li><li>从微流体装置的腔室除去固定液，用PBS洗涤等待10分钟，洗涤细胞三次。 </li><li>通过添加0.1％的Triton X-100的稀释剂，清洗剂通透细胞泰德在PBS中5分钟，以全部水库。 </li><li>从所有储层除去溶液和洗涤用PBS。重复洗涤3次等待洗涤之间10分钟。 </li><li>去除所有储层的溶液，并阻断含10％正常驴血清和3％无蛋白酶和免疫球蛋白G（IgG）的-free BSA的PBS缓冲液中孵育至少30分钟，在RT。 </li><li>旋转一次抗体原液以20,000×g离心5分钟以除去沉淀物。稀释抗体至适当的浓度在封闭缓冲液。取代从微流体装置中的封闭缓冲液与抗体溶液。样品孵育2小时，在室温或O / N在4℃。 </li><li>从所有水塘取出溶液并洗涤三次，用PBS，等待洗涤之间10分钟。 </li><li>旋转二次抗体原液以20,000×g离心5分钟以除去沉淀物。稀释抗体至适当的浓度在封闭缓冲液。更换PBS中的第二抗体溶液。孵化样品1小时，在室温。 </li><li>从所有水塘取出溶液并洗涤三次，用PBS，等待洗涤之间10分钟。 </li><li>通过直接吹打安装介质插入连接储存器的通道的开口代替PBS中以〜10-20微升的安装平台。让安装介质干燥〜20分钟 - 1小时RT。 </li><li>内染色两天（不超过1周），避光，以避免光漂白店设备在4℃下，并且图像轴突。 </li><li>染色完毕后，装入盖玻片与微流体装置上生长的显微镜载物台的海马神经元。定位染轴突成像在步骤4.1.1-4.1.7的区域。 </li><li>为三个信道的获取Z堆叠图像（300纳米步骤）:货物，电动机1，电动机2，采用一个高分辨率的目标（ 例如，具有高数值孔径的油或水的目标）。 注:收购​​Z-栈轴突往往不会长出完全平坦很重要在微通道中，因此，不是所有的荧光泪点都在同一焦平面上。 </li></ol></li><li>货运映射<ol><li>生成使用图像分析软件货物运输的kymograph。在ImageJ的插件通过Rietdorf和塞茨（EMBL）开发的建议产生kymographs（下载见 材料清单）。 注:ImageJ的是卫生27,28的国家机构制定了公共领域的，基于Java的图像处理程序（下载见材料清单）。 </li><li>通过重叠它们放到在kymograph的轨迹的位置处的固定点（ 图2A，B）中，使用市售的制图软件程序（材料对齐荧光货物（在步骤4.2.14生成）手动的定影图像列表）。手动通过第一重叠固定货物图像转印到kymograph和手动选择对应于特定轨迹上的货物泪点对准该kymograph。为了获得最好的比对结果，使用Z片是在映射的货物最佳焦点。几个ž片都可以使用。 </li><li>在Z堆叠的每个图像（货物，电机1和电机2;在步骤4.2.14产生）确定的XY坐标，并使用已建立的算法强度振幅对每个荧光泪点，以适合二维高斯函数的点扩散函数，它表示每一个点源在三个通道16,20,21（ 图2D）的。 注:要获得XY坐标，一个定制的MATLAB软件包被称为"电机共定位"开发16,20，其中纳入Jaqaman，Danuser和他的同事开发出21-23高斯拟合算法。根据要求可以得到其使用的软件和详细的说明。 </li><li>对于每个单独的货物泪点的分别映射到上kymograph的移动或固定的轨迹，选择吨他XY从"汽车共定位"计划的结果坐标和强度幅值（这些货物分别标示在图2B）。使用）在步骤4.2.14收购了几家Z堆栈的图像映射上找到不同的焦平面更多的货物。 </li><li>比较各个XY坐标的映射货物的那些方法每个马达通道中的对应的Z个切片（ 图2D）中获得。确定和选择在XY马达的坐标的货物的300nm的半径范围内的。对于货物，并且具有相同的焦平面内的信号，电机，使用"汽车共定位"软件包，以确定位于300纳米范围内的泪点。 </li></ol></li></ol>

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The authors have nothing to disclose.

这里介绍的协议可以实现个别荧光微管为基础的移动货物粒子运动的方向性，相对的类型和相关联的马达蛋白的量在活的神经元的相关性。此前，轴索膜泡货物的总电机组成测定在生化纯化小泡和细胞器9,15不同的人群。然而，对于一个单一类型的货物，在细胞内表征马达组合物一直是由于在纯化均一的囊泡种群的困难的挑战。此外，虽然电机堵转，力量测量果蝇</e...

胞内运输中的输送蛋白，膜，细胞器，和信号转导分子的各种蜂窝结构域1中的所有细胞类型是至关重要的。神经元是高度专业化与关键取决于必要的货物为他们的长途输送到各微区轴突胞内运输长，极化预测细胞。两个大家族的分子马达蛋白 - - 这种传输是通过驱动蛋白和动力蛋白介导的结合，以货物和沿顺行和逆行方向偏振的微管轨道上。而逆行运动由动力蛋白主要介导，在顺行方向上的移动是通过驱动蛋白马达的大，功能多样家庭容易。因此，轴突货物的顺行传输可以由驱动蛋白超家族1-5中各个家庭成员介导。虽然有些货物移动持续在任一方向，米OST货物移动双向，经常逆转的路上，他们的最终目的地1,5-13。此外，已经显示，同时相对的方向性关联到的货物，提高问题是，如何货物的调节的运动是由极性反转马达5-7协调的电动机。一起，轴突货物的运输是受电动机及其特定生化活性，而这又取决于各种适配器和监管的结合配偶体14的组合物调节的协同过程。 忠实地描述了轴突运输的机制为特定货物和揭开该传输的基本规则，它是最重要的，以确定他们的现场运输过程中与各个货物相关联的电动机蛋白及其调控结合配偶体的组合物。其他的方法，例如生物化学方法，提供平均MOT的估计或成分的纯化异构囊泡的人口，但这些估计不会透露的类型或关联到单个囊泡移动电机的数量。另外，沿着在体外预组装的微管泡运输的重组使测量在单个囊泡级15的一种类型的电机的数量。然而，这些实验并没有直接关联的电机与囊泡运输特性的量，并且在不存在细胞调节因子测量的传输。 的协议在这里提出，它决定了从个人移动​​小泡从免疫荧光（IF）的数据测量内源表达的马达蛋白马达组成（类型和马达的相对量），并关联这些参数完全相同的囊泡中的神经元的实时传输16。此方法需要IF生存货物运输数据进行精确的映射。这是通过慢慢生长完成克小鼠海马神经元微流体装置按照既定协议17-19。这些设备允许轴突和单个移动货物在固定和活光镜模式的识别和相关 （"映射"）（ 图1）。培养的神经元被转染了荧光标记的货物蛋白的运输被成像在高空间和时间分辨率，以获得该绘制在kymographs详细运动信息。在成像过程中，神经元是多聚甲醛固定，并随后用抗内源性马达蛋白。固定货物和电机的图像叠加到现场运动kymographs到"地图"（共定位），他们活货运动轨迹16。关联货物与马达蛋白的关联的现场动作，共定位是利用所谓的"莫一个定制的MATLAB软件包进行共定位器"16,20。荧光标记的货物和电动机产生衍射限制的点状的功能，可以部分地重叠。为解决重叠泪点的位置时，软件首先自动适合高斯函数对每个点扩散函数，表示各个荧光泪点，以确定其精确的XY子像素位置的坐标和强度振幅21-23电机和货物的位置是随后相互比较，以确定共定位16,20。因此，这种方法比其它方法24更精确地分配荧光泪点之间的共定位。 这种方法的优点是，以评估在固定的细胞马达与个别货物的共定位的能力，为此，活运动轨迹（例如，方向，其中将其在定影时的移动）已录制orded。用这种方法，驱动蛋白和动力蛋白被发现同时关联到该携带正常型朊病毒蛋白（的PrP ^ c -cellular），一个neuronally富集货物该移动双向或保持静止，在轴突16囊泡。该分析允许的工作模式制定了朊病毒Ç囊泡运动的调控，其中顺行（驱动蛋白）和逆行（动力蛋白）电机协调其活动，以移动囊泡在任一方向或保持静止，而相关的货物。这种方法的另一个优势是用于表征多，在几乎所有的细胞类型中移动，与所关注的任何其它蛋白（多个）荧光标记的货物的共定位/关联的潜在广泛的适用性。因此，活/固定关联可能潜在地允许对瞬时蛋白货物的相互作用的检测，因为许多个别的荧光标记的移动粒子可以在一个预定p被分析eriod时间。由于广泛的适用性和问题，该方法可以解决的类型，此协议将有兴趣的广大观​​众细胞生物学家包括学习贩运和运输中的神经元或其他类型的细胞。

<table><tbody><tr><td>poly-L-lysine</td><td>Sigma</td><td>P5899-20mg</td><td></td></tr><tr><td>D-MEM (Dulbecco&amp;rsquo;s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1x)</td><td>Life Technologies</td><td>11965092</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Neurobasal-A Medium (1x)</td><td>Life Technologies</td><td>10888022</td><td></td></tr><tr><td>B-27 Serum Free Supplement</td><td>Life Technologies</td><td>10888022</td><td></td></tr><tr><td>GlutaMAX I Supplement (100x)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>HBSS (1x) (Hank&amp;rsquo;s Balanced Salt Solution)</td><td>Life Technologies</td><td>24020117</td><td></td></tr><tr><td>DPBS (Dulbecco&amp;rsquo;s Phosphate Buffered Saline, no Magnesium, no Calcium (1x)</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Corning cellgro Water for Cell Culture&amp;nbsp;</td><td>Fisher Scientific</td><td>MT46000CM</td><td></td></tr><tr><td>Papain</td><td>USB Corporation</td><td>19925</td><td></td></tr><tr><td>DL-cysteine HCl</td><td>Sigma-Aldrich</td><td>C9768</td><td></td></tr><tr><td>BSA (bovine serum albumin)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G6152</td><td></td></tr><tr><td>DNAse I grade II</td><td>Roche Applied Sciences</td><td>10104159001</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>&amp;nbsp;11668027</td><td></td></tr><tr><td>Formaldehyde Solution 16% EM Grade</td><td>Fisher Scientific</td><td>50980487</td><td>Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations.</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H-3375</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-0121</td><td></td></tr><tr><td>BSA fatty acid and IgG free</td><td>Jackson Immuno Research</td><td>001-000-162</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>BP2403-4</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818-100</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36934</td><td></td></tr><tr><td>Cover Glass 1 1/2. 24 x 40 mm</td><td>Corning</td><td>2980-244</td><td></td></tr><tr><td>Axis microfluidic device, 450 &amp;micro;m</td><td>Millipore</td><td>AX450</td><td></td></tr><tr><td>Adobe Photoshop</td><td>Adobe</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-U&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Coolsnap HQ camera&amp;nbsp;</td><td>Roper Scientific</td><td></td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Fisher Scientific</td><td>12-565-95</td><td></td></tr><tr><td>150 mm cell culture dish</td><td>Fisher Scientific</td><td>12-565-100</td><td></td></tr><tr><td>Antibodies Used:</td><td></td><td></td><td></td></tr><tr><td>Anti-Kinesin light chain, V-17</td><td>Santa Cruz&amp;nbsp;</td><td>sc-13362</td><td>specificity verified using KLC1-/- neurons (Ref. 16), recommended dilution 1:100.</td></tr><tr><td>Anti Dynein Heavy Chain 1, R-325</td><td>Santa Cruz&amp;nbsp;</td><td>sc-9115</td><td>specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100</td></tr><tr><td>Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td><td>Life Technologies</td><td>A10042</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life Technologies</td><td>A31573</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody</td><td>Life Technologies</td><td>A11057</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody</td><td>Life Technologies</td><td>A21447</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td colspan="4">Plugins and Macros</td></tr><tr><td>ImageJ</td><td></td><td></td><td>&lt;a href=&quot;http://imagej.nih.gov/ij/index.html&quot;&gt;&amp;nbsp;http://imagej.nih.gov/ij/index.html.&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>ImageJ Kymograph Plugin</td><td></td><td></td><td>&lt;a href=&quot;http://www.embl.de/eamnet/html/body_kymograph.html&quot;&gt;http://www.embl.de/eamnet/html/body_kymograph.html.&lt;/a&gt;</td></tr></tbody></table>

characterizing the composition of molecular motors on moving axonal cargo using "cargo mapping" analysis

了解了这些分子马达协调其活动，以神经元内运输小泡货物的机制，要求电机/货运协会在单囊泡水平的定量分析。此协议的目标是使用定量荧光显微术来关联（"图"）的活的货物移动的位置和方向，以具有相同的货物相关联的马达的组成和相对量。 "货物映射"由荧光标记的货物移动在轴突上的微流体装置中培养的实时成像，随后通过化学固定记录的活运动期间，并在完全相同的轴突区域的随后的免疫荧光（IF）染色用抗马达。货物及其关联的电动机之间的共定位是通过分配的子像素位置的评估坐标电机和货物的通道，通过拟合高斯函数的衍射利mited点扩散函数表示各个荧光点源。固定货物和电机影像随后被叠加到地块货运动，以"图"到他们的跟踪轨迹。此协议的强度是活的组合和IF数据记录囊泡货物都在活细胞中的转运，并确定相关于这些完全相同的囊泡的电动机。该技术克服了使用生物化学方法来确定纯化的异构散装囊泡群体的平均电动机组合物，因为这些方法不露出于单个移动货物组合物前面的挑战。此外，该协议可以适于在其他细胞类型中的其他交通工具和/或运输途径的分析来关联个体的细胞内结构的运动与他们的蛋白质组合物。该协议的限制是相对较低的吞吐量，由于培养的低转染效率原代神经元和视可用于高分辨率成像的有限字段。未来的应用程序可以包括的方法，以增加神经元表达荧光标记的货物的数量。

图1示出了微流体装置的用于生长的海马神经元的概览（ 图1A，B）。神经元接种于容器1的微通道的尺寸可以防止细胞体（胞体）扩散到轴突隔室，而通道的长度可以防止树枝状突起从一路穿越到轴突隔室。后在培养〜2-3天，神经元开始穿过微通道延伸它们的轴突到轴突隔室（ 图1B中的C）。转染的轴突表达标记的黄色荧光蛋白（YFP-的PrP C）的正常型朊...

Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

表征分子马达的组成对运动轴突货物用"货物映射"分析

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

characterizing-composition-molecular-motors-on-moving-axonal-cargo

Research

JoVE Journal

Neuroscience

9.3K 次观看.  The Scripps Research Institute. Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

视频: Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

科研

生物化学

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

货物装载到驱动蛋白供电分子梭

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

生物学

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

神经科学

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

扩展轴突转运体内成像工具包

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

使用荧光显微镜驱动蛋白-1的货物的识别

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

表征分子马达的组成对运动轴突货物用"货物映射"分析

摘要

探索更多视频

此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

货物装载到驱动蛋白供电分子梭

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

切除小鼠神经神经纤维神经丝的成像与分析

扩展轴突转运体内成像工具包

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

使用荧光显微镜驱动蛋白-1的货物的识别

表征分子马达的组成对运动轴突货物用"货物映射"分析

摘要

探索更多视频

此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

货物装载到驱动蛋白供电分子梭

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

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