表征分子马达的组成对运动轴突货物用“货物映射”分析
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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 1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.
Materials
| Reagent and Equipment Name | Company | Catalogue Number | Comments |
| poly-L-lysine | Sigma | P5899-20mg | |
| D-MEM (Dulbecco’s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1X) | Life Technologies | 11965092 | |
| FBS (Fetal Bovine Serum) | Life Technologies | 10082147 | |
| Neurobasal-A Medium (1X) | Life Technologies | 10888022 | |
| B-27 Serum Free Supplement | Life Technologies | 10888022 | |
| GlutaMAX I Supplement (100X) | Life Technologies | 35050061 | |
| HBSS (1X) (Hank’s Balanced Salt Solution) | Life Technologies | 24020117 | |
| DPBS (Dulbecco’s Phosphate Buffered Saline, no Magnesium, no Calcium (1X) | Life Technologies | 14190250 | |
| Penicillin/Streptomycin (100X) | Life Technologies | 15140122 | |
| Corning cellgro Water for Cell Culture | Fisher Scientific | MT46000CM | |
| Papain | USB Corporation | 19925 | |
| DL-cysteine HCl | Sigma-Aldrich | C9768 | |
| BSA (bovine serum albumin) | Sigma-Aldrich | A7906 | |
| D-glucose | Sigma-Aldrich | G6152 | |
| DNAse I grade II | Roche Applied Sciences | 10104159001 | |
| Lipofectamine 2000 | Life Technologies | 11668027 | |
| Formaldehyde Solution 16% EM Grade | Fisher Scientific | 50980487 | Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations. |
| Sucrose | Fisher Scientific | S5-500 | |
| HEPES | Sigma | H-3375 | |
| Normal Donkey Serum | Jackson Immuno Research | 017-000-0121 | |
| BSA fatty acid and IgG free | Jackson Immuno Research | 001-000-162 | |
| Acetone | Fisher Scientific | BP2403-4 | |
| Ethanol | Fisher Scientific | BP2818-100 | |
| ProLong Gold antifade reagent | Life Technologies | P36934 | |
| Cover Glass 1 1/2. 24X40mm | Corning | 2980-244 | |
| Axis microfluidic device, 450 µm | Millipore | AX450 | |
| Adobe Photoshop | Adobe | N/A | |
| Nikon Eclipse TE2000-U | Nikon | N/A | |
| Coolsnap HQ camera | Roper Scientific | N/A | |
| 60 mm cell culture dish | Fisher Scientific | 12-565-95 | |
| 150 mm cell culture dish | Fisher Scientific | 12-565-100 | |
| Antibodies Used: | |||
| Anti-Kinesin light chain, V-17 | Santa Cruz | sc-13362 | specificity verified using KLC1-/- neurons (Ref. 16 ), recommended dilution 1:100. |
| Anti Dynein Heavy Chain 1, R-325 | Santa Cruz | sc-9115 | specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100 |
| Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody | Life Technologies | A10042 | recommended dilution 1:200. |
| Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody | Life Technologies | A31573 | recommended dilution 1:200. |
| Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody | Life Technologies | A11057 | recommended dilution 1:200. |
| Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody | Life Technologies | A21447 | recommended dilution 1:200. |
| Plugins and Macros | |||
| ImageJ | http://imagej.nih.gov/ij/index.html. | ||
| ImageJ Kymoraph Plugin | http://www.embl.de/eamnet/html/body_kymograph.html. |
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