Name: micromanipulation techniques allowing analysis of morphogenetic dynamics and turnover of cytoskeletal regulators
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作者没有什么可透露的。

在这里, 我们讨论了本文所描述的技术中的关键步骤, 以及如何在不同的实验条件下优化它们的应用。
显微注射是一种方法, 可用于监测细胞的即时效应, 从引进外源蛋白, 抑制剂, 或药物。它可以特别有利于确定蛋白质的功能在困难的染细胞类型或在情况下, 长期表达不需要。必须指出的是, 某些细胞类型的存活取决于它们所播种的细胞外基质。大多数内皮, 上皮, 或成纤维细胞类, 甚至小的像鱼基质 (见当et 等人21和安德森和交叉22) 可以成功地注入。然而, 也有一些例外, 例如在层粘连蛋白上播种的 B16-F1 细胞, 它构成了一个优良的细胞迁移模型系统, 但与这种类型的基质注射不相容, 原因不明。对于 NIH3T3 成纤维细胞, 我们通常在纤维粘连蛋白基底上进行注射, 以及其他 photomanipulation 技术, 如酶 (即使有 photoactivation; 在此显示的 B16-F1 细胞) 可以同样良好地执行在这些成纤维细胞 (见例如,Köstler et 。3). 还必须考虑到, 不同的蛋白质, 根据其功能特性和实验目标, 可能需要不同的时间来引起变化, 从几秒钟到几小时不等。该技术的优点是, 在使用质粒转染时, 在单细胞水平比例如更精确地控制外源剂的用量/浓度。此外, 荧光标记的蛋白质是不必要的, 以保证其存在的细胞, 这可以增加灵活性, 如果同时多通道可视化其他荧光标签的蛋白质是必需的。显微注射对于分析特定蛋白质或蛋白质混合物对细胞形态或细胞骨架的动态变化的即时影响特别有用 (例如,. 21 Arp2/3 复合抑制剂 Arpin 对迁移的即时影响示例)。技术的缺点是它的侵袭性, 可能导致细胞损伤或影响细胞形态学。因此, 在执行 microinjections 时, 一个重要的考虑因素是监测细胞的生存能力。此处介绍的方法依赖于手动操作。在测试的条件, 以配合成功的注射, 如成纤维细胞生长在纤维连接层, 这里所描述的手动注射协议允许近100% 成功率;这是必要的, 当结合这种方法与复杂和耗时的后续实验, 包括视频显微镜或酶, 如以前发布的3。这并不排除偶尔, 个别细胞可能会遭受微注射事件, 这可以被安全地识别的突然变化, 细胞核和细胞质的对比, 其次是细胞边缘退缩。这样罕见的实验性案例被排除并且因而不考虑为进一步分析。
然而, 半自动方法也通常被使用, 例如使用快速 (< 300 ms) 机器控制的针降低与射入压力增量重合, 因此针只必须在每个细胞之上被安置在各自之前注射。半自动注射的成功率低于上面描述的人工方法, 这仅仅是因为它对速度进行了优化, 接着分析了成功地在这种治疗中幸存下来的多个细胞;因而它不依靠成功的射入单独细胞。因此, 相对于单细胞分析, 半自动注射更适合分析数个百细胞的注射效果,例如,通过视频显微镜在低放大率或在细胞固定和染色。无论采用何种详细的方法, 显微注射都不构成终点化验, 但可以结合多种技术, 包括酶或 photoactivation3。
当通过酶确定蛋白质周转率时, 必须优化激光强度, 具体取决于显微镜的设置和成像条件 (放大、目标、等, 以及细胞类型、结构和荧光蛋白等。漂白)。请注意, 在最佳激光功率下, 高效漂白与最小可能的光损伤相结合, 以避免在分析 (例如, lamellipodia 或丝状伪足) 中的结构收缩或完全收缩, 甚至在细胞层面上造成损害。理想的情况是, 至少70–80% 的漂白效率应该达到, 虽然完全漂白可能会受到极快的蛋白质周转的阻碍, 在这种情况下, 任何50% 以上的东西也可以接受。对给定结构和荧光染料的最佳漂白功率应进行实验测试, 从低激光功率开始, 然后逐渐增加。当然, 任何荧光染料可以根据定义被漂白与激光接近其峰值的励磁 (488 毫微米为常用的绿色染料, 如 FITC 或 EGFP)。然而, 波长较短的激光, 如近紫外激光器, 提供了更高的功率, 因此也可用于有效漂白常用染料。我们通常使用405纳米二极管激光器 (120 兆瓦) 漂白的 EGFP 和红色荧光染料 (如 mCherry), 虽然其效率略低的情况下 (不显示数据)。由于405纳米二极管也可以用于 photoactivation (见下文), 它赋予这个系统最大的灵活性。
对于 B16-F1 细胞结构和荧光蛋白 photobleached, 在65–100兆瓦之间应用了405纳米激光功率。在分析 photobleached 区域时, 重要的是要考虑给定的结构是否保留在其原始形状上。例如, 在分析 lamellipodia 尖端蛋白质的周转时, 应注意 lamellipodia 的曲率是否随着时间的推移而显著改变, 因为如果分析的区域/轮廓不完全包含整个结构在每个被测量的框架。此外, 应该指出, 嵌入到 lamellipodia 中的捆绑, 如 microspikes, 可能会导致荧光强度的偏差。如图 2b (在9的时间帧中为白色箭头) 所示, 一个类似 microspike 的结构位于测量的 photobleached 区域的旁边, 但在整个测量期间仍处于外部, 因此不会导致任何误差。对于蛋白质周转的分析, 在选择分析区域的位置和大小时, 重要的考虑因素是它们的荧光随时间变化不应受到细胞形态学或其它因素的改变的显著影响, 而不是很难避免购置漂白。例如, 对所分析结构提供大量定量贡献的结构, 在分析过程中不应脱离实测区域;此外, 不相关的, 荧光实体, 如水泡结构, 吸引蛋白质不应该进入兴趣领域的分析。为确定 lamellipodial 肌动蛋白聚合的速率, 应注意不要对缩回或梳理 (即,向上折叠) lamellipodia 进行分析, 因为这将强烈影响结果的准确性。此外, lamellipodial 地区的退缩可能会出现快速向后移位, 有可能导致高估 lamellipodial 肌动蛋白聚合速率。另外考虑的是细胞内正常化区域的距离 (作为获取漂白校正的参考位置) 从漂白的实际位置, 应该足够大, 以避免直接photobleached 地区的影响。
在建立 photoactivation 绿色荧光标记结构的最佳条件时, 应注意避免 photoactivation 中的瞬时漂白。在我们的工作中, 获得的最佳结果是激光功率低于通常使用的 EGFP 漂白的5-10 倍。对于光活化作用分子的图像获取, 应通过考虑要光活化作用和分析的区域和结构的大小以及光活化作用的潜在移动性来优化帧间的曝光时间和时间间隔。蛋白质到其他亚细胞位置。对于所有类型的荧光成像, 维持细胞的生存能力是至关重要的获得生理相关的结果。
原则上, 荧光蛋白 (如 mEos 或 Dronpa 变体) 的绿-红 photoconversion 是一种同样强大的方法, 跟随 lamellipodium 的亚细胞结构的动态和更替 (见 例如,Burnette et 。23). 后一种方法相对于 PA-GFP 的优势是在转换前后采用两种不同颜色的蛋白质动力学的可能性, 而不需要共同表达额外的红色荧光蛋白。然而, 在我们的初步实验中, 与 photoconverted 探针相比, 在 photoactivation 上实现的荧光信号的对比度变化和强度更大, 可能是由于绿色与红色的优越光谱特征。荧光探针 (未显示数据)。在任何情况下, 对肌动蛋白长丝周转的详细研究, 如 lamellipodia 或痘苗病毒诱导的肌动蛋白的尾巴迄今只发表了使用 PA-GFP 衍生物5,6,24。
在考虑哪一个细胞区域来分析以下 photoactivation 时, 应该考虑几个因素, 这是用这里所示的具体例子来讨论的 (在细胞质激活后, 在细胞边缘加入肌动蛋白单体), 但当然可以推断出各种类似的科学问题。首先, 当测量 cytosolically 光活化作用蛋白的 lamellipodial 合并率时, 例如, 在不同的实验条件下 (如Dimchev et 所示)。6), 胞浆区域的大小及其与 lamellipodial 边缘的距离应在实验组之间比较。同样重要的是, 当 photoactivating 胞浆区域时, 细胞厚度在靠近细胞核的位置更大。激活较厚的细胞区域可能导致更多的活化蛋白, 因为在细胞质中 homogenously 分布的蛋白质的分布。最后, 被激活的蛋白质的表达水平在个体细胞肯定是高度可变的。由于所有这些可变性的考虑, 重要的是要比较在细胞中其他地方的 cytosolically 活化蛋白相对于在特定区域激活后获得的总荧光的合并水平。
我们已经描述了如何使用显微注射作为一个工具, 以调查蛋白质对细胞形态学的影响, 并表明了这一点, 证明了有效诱导 lamellipodial 结构在 NIH3T3 成纤维细胞杏仁与小 GTPase Rac1。我们以前应用这一技术来干扰细胞中的 Arp2/3 功能杏仁与 C 终端 WCA 领域的疤痕/波3。杏仁细胞中的各种参数可以通过其他的化验方法来分析, 如酶或 photoactivation。我们描述了酶和 photoactivation 如何用于研究肌动蛋白单体的亚细胞动力学和流动性。我们的小组以前使用过酶5来调查定位到 lamellipodia 的蛋白质的更替情况, 如虚拟、Abi、cortactin、cofilin 和上限蛋白, 或用于阐明在存在中的病灶粘连中成分的更替情况。和没有 Rac 信号4。此外, 测量肌动蛋白聚合速率可以通过漂白 EGFP 标记的β肌动蛋白5来完成, 但存在替代方法。跟踪荧光不均匀性的实时细胞成像-兼容探针标记细胞肌动蛋白花丝,如Lifeact25, 也可以使用6, 26.这里的优点是可以避免β肌动蛋白的过度表达, 它能够增加细胞边缘突起和迁移, 从而可能干扰特定的化验或实验问题 (参见例如,凯奇et 。26;Peckham et 。27). 然而, Lifeact 探针的一个明显的缺点是它的快速的开/关动力学与肌动蛋白花丝结合, 因此, 在细胞中标记的肌动蛋白长丝结构的漂白只提供探针更替的信息,但不是肌动蛋白花丝的营业额, 它绑定25。以前使用的荧光不均匀性的跟踪6, 26 确实提供了一个实际的折衷, 类似于广泛使用的跟踪荧光斑点纳入丝状骨架结构 (请参见例如鲑鱼和酶28), 但可能不像 EGFP 标记的 F 肌动蛋白结构那样直接使用和精确。Photoactivation 已经被我们应用于估计单体肌动蛋白在凸 lamellipodia 中的比例, 以及它在细胞质的移动, 在实验性地调整胞浆 F 肌动蛋白水平6。该技术在检查从相对较大的区域 (如胞浆区域) 衍生的蛋白质的流动性和分布方面是有用的。然而, 研究从相对较小的光活化作用结构中提取的蛋白质的分布;例如,由于激活的荧光分子数量少, 信号微弱, 因而缺乏灵敏度, 因此生长锥可能具有挑战性。荧光 photoactivation 或 photoconversion 的潜在替代技术 (见上文) 可能包括逆酶, 它依赖于漂白整个细胞, 除了 ROI, 然后跟踪荧光分子的流动性远离这个地区。该技术不需要 overexpressing photoactivatable 版本的蛋白质, 但将始终涉及暴露在异常高剂量的激光功率, 可能导致不必要的副作用, 如光损伤。
显然, photoactivation 和酶不能区分蛋白质是否作为单体、脂肪酸、甚至小的低聚物移动, 以及它们是否与附加的结合性伙伴相结合。这类信息可以通过荧光相关光谱学技术 (29 ) 来获得, 或者是电影-烦恼30。尽管如此, 酶和 photoactivation 是直接评估细胞内局部和全球蛋白质动态的简单方法, 而不管感兴趣的蛋白质、亚细胞位置或研究的类型。

在细胞和分子生物学的许多领域中, 监测蛋白质和其他分子的时空动力学已经成为一个必不可少的工具。先进的荧光显微技术包括荧光共振能量转移 (烦恼) 和焦虑荧光寿命成像 (烦恼-电影), 或酶, 荧光损失漂白 (翻转) 和 photoactivation 以及许多其他允许对于蛋白质-蛋白质相互作用的时空跟踪, 构象的变化, 以及在细胞1,2中确定不同蛋白质的扩散和定位的动力学。酶和 photoactivation 技术, 特别是, 广泛适用于检查调节肌动蛋白细胞骨架和细胞迁移。这些技术可以单独应用或与附加的微操作系统技术 (如显微注射 3) 结合使用, 并涉及荧光标记蛋白的表达.它们允许将蛋白质联想的动力学估计为参与细胞迁移的富含肌动蛋白的结构, 如丝状伪足或 lamellipodia、病灶粘连中蛋白质的更替4或分支肌动蛋白网络 5.它们还能确定 lamellipodial 肌动蛋白聚合速率, 单体肌动蛋白在细胞质内的分散度, 亚细胞肌动蛋白单体易位率在突出lamellipodia6和其他参数。
酶是一种可视化和量化在活细胞内蛋白质流动性的方法, 最初在二十世纪七十年代由阿克塞尔罗德7开发。一个在细胞内的感兴趣区域 (ROI), 用荧光标记的蛋白质填充, 瞬时暴露在高强度的激光中, 足以导致在给定的短时间内对该区域中的荧光分子进行漂白。在漂白过程中, 未漂白的、荧光标记的蛋白质, 会根据它们的时空动力学, 扩散和渗透到漂白的区域, 从而导致 photobleached 分子随着时间的推移而迁移。漂白地区荧光回收率的大小取决于各种因素, 包括给定分子的扩散率和传播速率, 以及在假定相关的漂白结构中的更替率。因此, 可溶性蛋白质将通过扩散迅速地在漂白的 ROI 中调节荧光的恢复, 而与结构紧密相关的蛋白质, 如焦粘连, 将有更长的周转时间, 因为它们的荧光恢复将依赖于蛋白质可溶性分数的扩散和结构相关分数的离解-联想动力学。荧光恢复通常是获得和量化, 直到初始水平的前漂白强度的荧光达到。但是, 如果初始荧光强度的一部分属于所谓的不动分数, 则无法通过扩散补充或以非常慢的速率补充, 而与包括移动的大多数分子相比, 这种情况不会发生。分数。为了确定蛋白质更替率, 酶曲线的产生, 代表了荧光恢复的程度随着时间的推移。从这些恢复曲线可以计算蛋白质回收率的平均半倍。通过创建平均酶数据的曲线拟合, 进而进行数学分析, 也可以推断流动分数的平均周转率是否构成一个分子的均匀种群的组合, 或者它是否由两个或更多的分子亚群在不同的速率翻转。除了通过定量方法估计蛋白质周转率外, 跟踪 lamellipodia 中 photobleached 区域的恢复也可以精确量化 lamellipodial 运动参数, 如逆行流、突起、和肌动蛋白聚合速率。因此, 酶构成了一种多功能工具, 用于评估活细胞结构中的各种参数。
Photoactivation 是一种用于跟踪来自指定细胞位置的蛋白质或分子的扩散和流动性的方法。例如, 该技术采用了一种野生型绿色荧光蛋白 (GFP) 的变体, 最初由帕特森和利平科特8开发, 这种变异方式使其荧光在接触到紫外线 (紫外线) 光 (大约400毫微米; 这里, 405 毫微米)。正如帕特森et al所描述的, 野生型 GFP 染色体作为中性酚类和阴离子 phenolates 的混合种群存在, 其产生的吸光度峰值约为 397 nm, 较小的是 475 nm。在紫外光照射下, 种群经历 photoconversion, 向阴离子形态转移。当兴奋 488 nm, photoconverted/光活化作用蛋白表现出3倍的荧光增加, 在实践中缺乏区分激活和非活化 GFP 由于高内在背景荧光。然而, 通过在 GFP 序列中引入单一氨基酸突变 (在位置 203), 降低了背景强度。由此产生的 T203H 突变体, 也称为 photoactivatable-gfp (PA-gfp) 的特点是显著降低了小峰的吸光度, 这在辐照与紫外线照射后增加近100倍, 随后激发 488 nm 光。因此, 过度表达的 PA-GFP 标记蛋白是一种广泛使用的方法, 它允许测定分子在细胞内的扩散和运动。我们以前应用了 PA-GFP 标记肌动蛋白, 以确定肌动蛋白单体分散的速率从胞浆区域, 不仅允许探索他们的流动性在细胞质, 而且他们的合并率到突出lamellipodial 肌动蛋白网络6。最近的文献还介绍了新颖的, 可转换的蛋白质, 原则上可以使用类似的方式, 但窝藏潜在的优势已经可见, 在照片转换之前。这组荧光蛋白的例子包括 Dendra2 和 mEos29,10,11,12。
在本文中, 我们解释了 microinjecting 细胞与蛋白质的方法。我们进一步解释了这项技术如何与酶结合, 通过漂白蛋白参与肌动蛋白细胞骨架调节和运动, 以及如何酶曲线和半时间恢复的移动分数可以获得。此外, 我们提供了一个例子, 酶技术如何可以用来确定肌动蛋白聚合速率的 lamellipodial 网络。我们还提供有关如何执行 photoactivation 实验的指示和提示, 可用于确定单体肌动蛋白的胞浆流动性和肌动蛋白在 lamellipodia 中的比例。当然, 这些技术不仅限于跟踪肌动蛋白骨架成分, 而且在可能需要适度的适应或优化的情况下, 可以广泛应用于其他细胞类型或研究不同的蛋白质、结构和参数。

<table><tbody><tr><td>B16-F1 mouse skin melanoma cells&amp;nbsp;</td><td>American Type Culture Collection, Manassas, VA</td><td>CRL-6323</td><td></td></tr><tr><td>NIH-3T3 cells</td><td>American Type Culture Collection, Manassas, VA</td><td>CRL-1658</td><td></td></tr><tr><td>DMEM 4.5g/L glucose&amp;nbsp;</td><td>Life Technologies, Thermno Fisher Scientific, Germany</td><td>41965-039</td><td></td></tr><tr><td>Ham&amp;rsquo;s F-12 medium</td><td>Sigma-Aldrich</td><td>N8641</td><td></td></tr><tr><td>Fetal calf serum&amp;nbsp; (FCS)</td><td>PAA Laboratories, Linz, Austria</td><td>A15-102</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich, Germany</td><td>F7524</td><td>Lot054M3396</td></tr><tr><td>MEM Non essential amino acids&amp;nbsp;</td><td>Gibco, ThermoFisher Scientific, Germany</td><td>11140035</td><td></td></tr><tr><td>L-Glumatine 200mM (100x)</td><td>Life Technolgies</td><td>25030-024</td><td></td></tr><tr><td>Pen-Strep 5000 U/mL</td><td>Life technologies</td><td>15070063</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco, ThermoFisher Scientific, Germany</td><td>11360-039</td><td></td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L-2020</td><td></td></tr><tr><td>Laminin coating buffer&amp;nbsp;</td><td></td><td></td><td>Self-made: 50mM Tris ph7.4, 150mM NaCl</td></tr><tr><td>Fibronectin from human plasma&amp;nbsp;</td><td>Roche Diagnostics, Mannheim, Germany</td><td>11 051 407 001</td><td></td></tr><tr><td>Jetpei</td><td>Polyplus Transfection, Illkirch, France</td><td>101-10N</td><td></td></tr><tr><td>JetPei buffer</td><td>Polyplus Transfection, Illkirch, France</td><td>702-50</td><td>150mM NaCl</td></tr><tr><td>PA-GFP-actin plasmid DNA&amp;nbsp;</td><td></td><td></td><td>described in Koestler et al.2008</td></tr><tr><td>pEGFP-actin plasmid DNA</td><td>Clontech, Mountain View, CA, USA</td><td></td><td></td></tr><tr><td>Rac1 protein for microinjection</td><td></td><td></td><td>Purified as GST-tagged version, and cleaved from GST prior to injection</td></tr><tr><td>Microinjection buffer</td><td></td><td></td><td>Self-made: 100mM NaCl, 50mM Tris-HCl ph7.5, 5mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1mM DTT</td></tr><tr><td>Dextran, Texas Red, 70,000 MW, Lysine Fixable</td><td>Molecular Probes, Thermno Fisher Scientific, Germany</td><td>D1864</td><td></td></tr><tr><td>Microscope circular cover glasses 15mm, No.1&amp;nbsp;</td><td>Karl Hecht, Aisstent, Sondheim, Germany</td><td>1001/15</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader Tips</td><td>Eppendorf, Hamburg, Germany</td><td>5242 956 003</td><td></td></tr><tr><td>Eppendorf Femtotip Microinjection Capillary Tips</td><td>Eppendorf, Hamburg, Germany</td><td>930000035</td><td></td></tr><tr><td>Silicone Grease&amp;nbsp;</td><td>ACC Silicones, Bridgewater, England</td><td>SGM494</td><td></td></tr><tr><td>Aluminium Open Diamond Bath Imaging Chamber</td><td>Warner instruments</td><td>RC-26</td><td></td></tr><tr><td>Automatic temperature controller</td><td>Warner Instruments</td><td>TC-324B</td><td></td></tr><tr><td>Microscope: Axio Observer&amp;nbsp;</td><td>Carl Zeiss, Jena, Germany</td><td></td><td></td></tr><tr><td>CoolSnap-HQ2 camera&amp;nbsp;</td><td>Photometrics, Tucson, AZ</td><td></td><td></td></tr><tr><td>Lambda DG4 light source&amp;nbsp;</td><td>Sutter Instrucment, Novato, CA</td><td></td><td></td></tr><tr><td>Laser source&amp;nbsp;</td><td>Visitron Systems</td><td></td><td></td></tr><tr><td>Eppendorf FemtoJet microinjector&amp;nbsp;</td><td>Eppendorf, Hamburg, Germany</td><td></td><td>With built-in compressor for pressure supply</td></tr><tr><td>Nikon Narishige Micromanipulator system</td><td>Nikon Instruments, Japan</td><td></td><td></td></tr><tr><td>Visiview software v2.1.4</td><td>Visitron Systems, Puchheim, Germany</td><td></td><td></td></tr><tr><td>Metamorph software v7.8.10</td><td>Molecular Devices, Sunnyvale, CA</td><td></td><td></td></tr><tr><td>Sigma Plot v.12</td><td>Systat Software Inc.</td><td></td><td></td></tr></tbody></table>

micromanipulation techniques allowing analysis of morphogenetic dynamics and turnover of cytoskeletal regulators

研究蛋白质的时空动力学可以揭示它们在不同语境中的功能重要性。本文讨论了漂白 (酶) 和 photoactivation 技术后荧光恢复方法在亚细胞位置蛋白质时空动力学研究中的应用。我们还展示了这些技术如何能够直接确定与肌动蛋白骨架调节和细胞运动有关的各种参数。此外, 细胞的显微注射还被描述为一种替代疗法 (可能之前或补充上述 photomanipulation 技术), 以触发移位蛋白对细胞的瞬时影响形态和功能。微操作系统, 如蛋白质注射或局部应用血浆膜通透性药物或骨架抑制剂, 可以作为一个强有力的工具, 以记录特定治疗的直接后果的细胞行为在单细胞和亚细胞水平。这是在这里的例子, 立即诱导 lamellipodial 细胞边缘突出的注射重组 Rac1 蛋白, 建立了一个季度前。此外, 我们提供了一个协议, 以确定增强的绿色荧光蛋白 (EGFP)-虚拟的营业额, 肌动蛋白长丝聚合酶显著积累在 lamellipodial 提示 B16-F1 细胞, 使用酶和包括相关数据分析和曲线拟合。我们还提出了评估 lamellipodial 肌动蛋白网络聚合速率的指导原则, 如表达 EGFP 标记β肌动蛋白的细胞所示。最后, 给出了如何调查细胞胞浆内肌动蛋白单体流动率的指示, 其次是肌动蛋白在快速灯丝组装部位的 lamellipodia, 如突出的小贴士, 使用 photoactivation方法。这些协议都不限于肌动蛋白细胞骨架的成分或调节器, 但可以很容易地扩展到类似的方式探索蛋白质在不同亚细胞结构或功能中的时空动力学和功能上下文。

图 1g, h显示 NIH3T3 成纤维细胞的相对比图像, 前和10分钟后显微注射 Rac1, 这是一个小的细胞家族 GTPase 能够诱导 lamellipodia 形成通过与波复合体的相互作用。在微注射 (图 1g) 之前, 该单元格首先被可视化, 以确认其生存能力和形态学,例如,缺少 lamellipodia。在注射后10分钟后, 细胞已经明显改变了它的形态学, 这是预期从这种治疗, 并表示成功注射 (图 1h)。
为了简单和清晰起见, 我们接下来为细胞中的酶和 photoactivation 分析提供了模范的结果, 而这些研究还没有杏仁。
在图 2EGFP 中显示了在 lamellipodium 提示处标记的虚拟的营业额的分析.请注意, 虚拟另外目标到新生和焦点粘连, 小和拉长点在细胞内部18,19。lamellipodial 区域的荧光强度在尖端有明确的虚拟积累被漂白和测量每一个时间框架, 通过遵循 ROI 的轮廓之前, 期间, 漂白后, lamellipodium 突出向前。由于漂白的 EGFP-虚拟蛋白在这些部位被非漂白分子回收, 观察到荧光的逐渐恢复 (图 2b)。在图 2c中可以看到以这种方式获得的酶恢复曲线, 并将其规范化为前漂白强度 (表示为 1)。漂白效率可以变化, 约20% 的价值之前漂白在这个例子, 从价值确定从 t0 (第一帧后漂白)。荧光的增加到达一个高原在例子显示在大约80% 荧光在漂白之前。在静态结构中, 在实验的时间过程中, 如焦粘度, 恢复后达到的前漂白强度和高原荧光的差异定义为不动分数 (如果,图 2中的红色箭头)c、e), 而在漂白和完全恢复时间之间恢复的荧光量被定义为移动分数 (图 2c、e中的绿色双头箭头)。请注意, 在动态变化的结构, 如在这里分析的 lamellipodium 尖端, 其范围可能不仅代表不动分子, 但也从降低的凸出速度, 因为 EGFP 虚拟强度是众所周知的依赖于此参数18。要计算恢复的半时间, 在斯格码绘图 (图 2d) 上创建了一个拟合曲线。在这种情况下, 从解决等式 2中提取的 "b" 参数值等于 0.0754, 当应用到对数函数 (等式 4) 时, 将导致估计的恢复时间为9.19 秒 (图 2d, 远右面板), 在这个特定的单元格中相对较快, 这与以前发布的平均5相比。必须指出的是, 在同一人群中, 恢复半衰期有时会有很大差异。因此, 为了获得具有代表性的结果, 我们建议将此参数确定为至少15-20 个单元格的平均值。为了说明方差的程度, 生成了每个时间点平均从15个单元 EGFP 虚拟恢复的算术方法 (图 2e), 并以类似的方式创建和显示平均曲线匹配 (图 2f)。
lamellipodial 肌动蛋白网络的聚合速率包括正向网络凸出和逆行流的总和。酶可用于测量肌动蛋白聚合速率的转染细胞 (在这种情况下 B16-F1) 与 EGFP 标记β肌动蛋白和漂白一个突出的 lamellipodial 区域 (图2 g).为分析 lamellipodial 肌动蛋白网络聚合, EGFP 标记的β肌动蛋白漂白后的荧光恢复是随着时间的推移进行评估的。由于肌动蛋白单体的聚合进展在 lamellipodial 肌动蛋白花丝的有刺的末端 (所有指向前面20), 网络经常移位后方和进步批转, 速度可以容易地通过漂白上的荧光极化恢复获得。当漂白带到达 lamellipodium 后部与薄板的过渡区后, lamellipodium 的荧光恢复就完成了, 其特征是水平排列的纤维束密度较低。比 lamellipodium 中观察到的要慢得多。如图 2g所示, 荧光恢复可以被可视为水平线向边缘的直线, 向后流向薄板, 这允许测量凸出和逆行流的距离 (单独表示在图 2g的最右侧面板中, 分别为橙色和红色双头箭头。
我们还应用 photoactivation 在 B16-F1 细胞转染的 PA-GFP-肌动蛋白, 以跟踪肌动蛋白单体在细胞质的流动性和其在突出 lamellipodia 的比例。如图 3a、b所示, 一个胞浆区域通过暴露于 405 nm 激光光活化作用, 而在 gfp 通道上每1.5 秒获取图像, 以可视化 gfp 标记的光活化作用肌动蛋白的分布。光活化作用 GFP-肌动蛋白在图 3 b 中的胞浆区域中可见扩散.光活化作用胞浆区的荧光强度下降率表现为 t0 (photoactivation 后第一帧) 初始强度的百分比;图 3c). 光活化作用肌动蛋白也集成在 lamellipodia 的提示, 其中新的肌动蛋白单体添加到增长的刺状的两端拉长肌动蛋白花丝在突出。为了估计 lamellipodial 的速率, 我们测量了一个二维轮廓/区域约5µm 宽度和1µm 高度的荧光强度;该地区在 lamellipodium 的尖端不断被重新定位。肌动蛋白在 t0 (图 3d) 中代表了光活化作用胞浆区域的荧光强度百分比。随着肌动蛋白纤维的伸长, 新的肌动蛋白单体在 lamellipodial 前被纳入。这些肌动蛋白单体的一小部分是随机衍生的胞浆池, 其中单体被光活化作用。这导致 lamellipodia 在 photoactivation 后的第一二十年代, 荧光的快速增加。随着新的单体被添加到 lamellipodial 前面, 以前加入的肌动蛋白单体流动与花丝向薄板逆行流动。随着时间的推移, ROI 是完全充满了荧光单体和一个高原在荧光达到 (图 3d)。然后观察到, 当光活化作用单体在整个细胞中扩散后, 荧光的逐渐下降, 非光活化作用肌动蛋白单体越来越多地被重新添加到 lamellipodial 的前方。荧光的减少将发现一个新的高原, 当平衡在光活化作用和非光活化作用单体之间的整个细胞被到达 (没有显示的数据) 时, 将很快到达。
肌动蛋白单体在整个细胞质的流动性是通过测量在光活化作用区域远端位置相同大小的区域的荧光强度而得出的 (举例说明在图 3 a 上的颜色编码区域标记为R1-R5)。如图 3e所示, 这些区域中的荧光强度逐渐减少, cytosolically 光活化作用区域, 因为光活化作用肌动蛋白单体的分数变得越来越稀释非活化 (即,非荧光) 单体。此外, 荧光的峰值是在以后达到的: 测量的区域从光活化作用区域的距离越远, 肌动蛋白单体扩散到这些区域所需的时间就越长。通过对到达荧光高原的半时间进行量化, 可以得出一个代表肌动蛋白单体渗入每个区域的程度的代表性值。该区域越远, 光活化作用肌动蛋白扩散到它所需的时间越长, 因此要达到的荧光平台需要更多的时候, 最终导致更高的t 1/2 值(图 3 e).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57643/57643fig1.jpg"> 图 1: 成像室组件和显微注射过程.(a) 成像室组件。(b) 硅脂在塑料封口机的开口周围小心涂抹。(c) 盖玻片位于成像腔开口中心的单元侧。(d) 通过将塑料封口机放置在盖玻片的顶部并拧紧侧面夹具来建立安全密封。(e) 显微镜介质被 pipetted 到腔槽中。(f) 成像室位于显微镜阶段, 热检测器和电极连接到加热单元预设置为37°c, 并且允许细胞在显微镜开始前至少适应30分钟。在这个例子中, 显微镜阶段也配备了机器人执行 microinjections, 和注射针被浸入介质覆盖细胞层在成像室。(g) NIH3T3 成纤维细胞在显微注射前通过相衬显微术进行可视化。红十字在核周车厢表明未来微注射的地点, 对应于高细胞质区域由于接近大核。(h) 10 分钟后, 微注射与 Rac1, 细胞的反应由 lamellipodia 的显著形成周围的整个细胞周围 (由绿色箭头指示)。<a href="/files/ftp_upload/57643/57643fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57643/57643fig2.jpg"> 图 2:酶允许确定蛋白质周转率或 lamellipodial 肌动蛋白聚合.(a) 代表在 lamellipodial 区域漂白前表示 EGFP 虚拟的 B16-F1 细胞的典型例子。不同颜色的轮廓/形状被标记, 以表明哪些区域被考虑为荧光强度测量随着时间的推移。注意标记有感叹号的红色轮廓, 它标签的胞浆区域位于包含多个囊泡和细胞表面褶边的区域内。在选择荧光参考区域时应避免这样的动态区域, 因为它们的特点是荧光的短期波动很强, 可能导致不准确的结果。(b) EGFP 虚拟在漂白前后表达细胞的 Lamellipodial 区域。在紫色标记的区域内漂白后的荧光信号恢复是随着时间的推移而可视化的。箭头表示 microspike 的尖端, 虚拟可能由于肌动蛋白花丝聚合的高密度而富集19。(c) 酶恢复曲线的一个示例, 它从量化 photobleached lamellipodium (紫色轮廓) 的荧光强度到 b. 右侧红色和绿色线条分别表示不动和移动分数。(d) c (左面板) 中的酶恢复曲线的适当性, 以及用于派生恢复半时间 (右面板) 的计算方法的示例。(e) 以15个细胞的荧光恢复曲线为平均值的酶恢复曲线的一个例子, 用 SEM 条表示样本种群内的变异性程度。(f) 从平均酶恢复曲线适合15个单元格 (左面板) 的曲线拟合, 以及用于推导恢复半时间 (右面板) 的计算方法示例。(g) 显示 EGFP 标记β肌动蛋白的 B16-F1 细胞在 lamellipodial 区域漂白之前和之后 lamellipodium 的时间推移板, 随后在 lamellipodium 随着时间的推移荧光恢复。在最右边的面板上, 为凸出和逆行距离测量的值 (分别为橙色和红色)。在图像面板下进行计算, 揭示了 lamellipodial 肌动蛋白网络的聚合速率与凸出和逆行距离的总和。<a href="/files/ftp_upload/57643/57643fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57643/57643fig3.jpg"> 图 3:Photoactivation 在整个细胞内进行单体跟踪的 PA-GFP 肌动蛋白.(a) 一个代表 B16-F1 细胞的典型例子, 表达 pa-GFP 肌动蛋白, 在触发 photoactivation 之前, 在胞浆区域 (pa) 表示。不同颜色的轮廓标记, 以表明哪些区域被考虑为荧光强度测量随着时间的推移。(b) 说明 photoactivation 后的 PA-GFP 肌动蛋白的时间分布。注意在光活化作用、胞浆区 (红圈) 中荧光的逐渐减少, 因为光活化作用肌动蛋白扩散远离它。由于其扩散到前端和组装到网络, 光活化作用肌动蛋白单体逐渐增强, 在 lamellipodia (青色地区) 和整个细胞质 (不同的颜色编码区域) 的距离和时间依赖的方式。(c) 代表, 光活化作用胞浆区内荧光的时间下降 (b 中的红色轮廓)。(d) lamellipodial 区域荧光强度的时间变化 (b 中青色轮廓)。(e) 曲线代表了胞浆区域荧光强度的时间变化 (在 b 中的颜色编码), 因为定位于 photoactivation 区域的可变距离。注意到达荧光高原的一半时间 (在右侧的图例中表示) 随着给定区域与 photoactivation 区域的距离增加, 可能与肌动蛋白单体扩散所需的增加时间相关。各自区域。<a href="/files/ftp_upload/57643/57643fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators at JoVE.com

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

我们描述了微和 photomanipulation 技术, 如酶和 photoactivation 如何能够确定运动参数和蛋白质在迁移细胞内的时空动力学。实验读数包括亚细胞动力学和运动调节器的更替或底层肌动蛋白骨架。

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

micromanipulation-techniques-allowing-analysis-morphogenetic-dynamics

Universidad San Sebastian

Research

JoVE Journal

Biology

10.0K 次观看.  Technische Universität Braunschweig. 我们描述了微和 photomanipulation 技术, 如酶和 photoactivation 如何能够确定运动参数和蛋白质在迁移细胞内的时空动力学。实验读数包括亚细胞动力学和运动调节器的更替或底层肌动蛋白骨架。

视频: Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

科研

生物工程

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遗传学

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

发育生物学

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

一种微流体方法，用于控制体力生成的信号振荡的功能研究

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

生物学

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented.

使用差分动力学显微镜定量细胞骨架动力学

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

神经科学

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

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 ...

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

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

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

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

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