Nanopodia - 薄，易碎膜预测与细胞运动和细胞间的相互作用角色
Summary
Nanopodia很瘦但很脆弱的膜通道，向上延伸至100微米的细胞的领先的前面或后面尾随和检测的细胞环境。直接固定在较高的Triton X-100浓度为37℃，轻柔洗涤，并避免如乙醇，甲醇，丙酮或有机溶剂，并须遵守这些细胞结构。
Abstract
贴壁培养细胞保持极化状态，支持移动和细胞间的相互作用。 Nanopodia是薄的，细长的，主要是丝状肌动蛋白阴性的内皮细胞和癌细胞膜突起，可以通过TM4SF1（跨膜-4-L-6家族-1）的免疫荧光染色进行可视化。 TM4SF1集群在100-300微米直径TMED（TM 4SF1Énriched微Ðomains）含有3到多达14个独立的TM4SF1分子。 TMED间歇地沿着nanopodia以1至3％μ米TMED并牢固地固定到nanopodia矩阵具有规则间距。这使得nanopodia从偏振内皮细胞或肿瘤细胞的前前或后后方延伸超过100μm，且使膜的残基上MATR被留下九时电池移开。 TMED和nanopodia被忽视了，因为他们的极端脆弱性和敏感性，以温度。常规洗涤和固定破坏的结构。 Nanopodia被直接固定在多聚甲醛（PFA）在37℃下保存，然后短暂暴露于0.01％的Triton X-100的染色之前。 Nanopodia在细胞生物学中打开新的局面：他们承诺将重塑我们的细胞如何感知他们的环境的认识，检测和识别其他细胞的距离，启动细胞间相互作用的密切联系，以及参与运动，增殖的信号传导机制，以及细胞细胞通讯。被用于研究TM4SF1衍生nanopodia开发的方法可以是用于通过经典四旋蛋白，特别是广泛表达CD9，CD81，和CD151的机构形成在其它类型的细胞nanopodia的研究是有用的。
Introduction
在极化运动，动物细胞延长了多种从它们的表面活力，细胞膜突起，其中包括丝状伪足，伪足，回缩纤维，和褶边1。最近加入到这个名单是nanopodia，一个新认识的类型的薄（100-300微米，直径），伸长（可达50〜100微米长）投影膜提供的膜通道的F-肌动蛋白的结构，如丝状伪足的延伸和回缩纤维，而且染色水平与TM4SF1（跨膜-4-L-6家族-1）中培养的内皮细胞和肿瘤细胞2,3。
TM4SF1是用四旋状的拓扑结构，最初被称为发现，即该分子是内皮细胞的生物标记物中起着内皮细胞增殖和迁移2,3必不可少的作用前的肿瘤细胞抗原4的蛋白质。免疫荧光染色发现TM4SF1被定位于perinuc利尔囊泡和到质膜，并富含TM 4SF1ënriched微ðomains（TMED）。 TMED锚nanopodia到矩阵和存在于nanopodia 1-3 TMED /微米长度的规则排列的条带状花纹。 Nanopodia通常是从一个细胞的前领先，并延长电池的极化为移动中尾随后面。由于TMED的牢固附着的性质，nanopodia无法缩回到细胞，因为它移动远离;弃nanopodia残基从而描绘出蜂窝移动路径。 Nanopodia提供的膜通道的F-肌动蛋白的扩展和回缩，且细胞间的相互作用和通信2,3的网站。这些特点意味着nanopodia提供研究细胞分化，对环境的感知细胞，测定细胞运动的路径和方向，和细胞间的相互作用过程中的F-肌动蛋白装配背后的机制一个独特的机会，一个ND通信。
由于TMED的高度疏水性质和nanopodia的薄和脆弱的膜性质，需要特别注意采取以保存TMED和nanopodia。 nanopodia和去除TMED由共同的实验室方法的破坏是一个可能的原因，只有63出版物自1986年1它的首次发现和完全缺乏TM4SF1富集在100-300微米微区上的知识出现在TM4SF1细胞表面，直到TM4SF1的内皮细胞在2009年2报告。
常规的免疫染色方法通常使用像乙醇，甲醇，丙酮或有机溶剂来修复细胞，并使用0.1％或更高的Triton X-100浓度通透细胞5。这里所描述的研究实现了三个重大变化，以常规方法揭示TMED和nanopodia：（i）使用37℃的4％PFA和修复细胞的3776，C培养箱，（ⅱ）申请温和室温下PBS中洗涤，并且（iii）使用低于0.03％的Triton X-100只简要地通透细胞除第一抗体之前，如曲通X-100 0.03％更高将提取TM4SF1。
所有四旋蛋白形成细胞膜上的6微区和在内皮细胞和肿瘤细胞中某些2,3共定位与TMED。如像CD9，CD81，和CD151四旋蛋白被广泛表达，在这里所描述的染色方案可以扩展到那些缺乏TM4SF1对nanopodia功能的研究很多不同的细胞类型。
Protocol
Representative Results
Discussion
Nanopodia薄细胞膜通道，牢牢附着基质通过TMED，可以从一个极化移动细胞感知环境并介导细胞间的相互作用2,3延伸超过100微米。 Nanopodia坚持以矩阵如此坚定地认为残留物留下的细胞移开（ 图1和图2）。 Nanopodia从而使我们能够研究细胞如何感知他们的环境，并确定细胞运动的路径，基因表达或药物治疗是如何变化的运动。然而，由于这些薄（100-300微米宽度）膜结?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们承认哈罗德·德沃夏克博士的有益讨论包括建议尽量固定在37℃煤灰。这项工作是由美国国立卫生研究院授予P01 CA92644和由国家癌症研究基金会的合同支持。
Materials
| glass discs | Fisher Scientific | 12-545-82 | 12-mm diameter |
| glass jar | Fisher Scientific | 02-912-310 | Certified Clean Clear Glass Straight-Sided Jars, 4 oz. |
| glass slide | Fisher Scientific | 12-544-1 | Fisherfinest Premium Plain Glass Microscope Slides |
| 50-ml falcon tube | BD Falcon | 352070 | 50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile. |
| 15-ml falcon tube with Styrofoam rack | Corning | 430790 | Corning 15-ml PP Centrifuge Tubes |
| Sharp forceps | Fisher Scientific | 13-812-42 | Dissecting Extra-Fine-Pointed Splinter Forceps |
| 24-well cell culture plate | BD Bioscience | 353047 | 24-well Cell Culture Plate |
| 150-mm cell culture plate | BD Bioscience | 353025 | 150-mm cell culture plate |
| 19G 1 ½ syringe needle | BD Bioscience | 309635 | 19G 1 ½ |
| Name of Reagent | |||
| ethanol | Decon Laboratories, Inc. | 2701 | Decon's Pure Ethanol 200 Proof |
| collagen solution | BD Bioscience | 354249 | Collagen I, High Concentration, rat tail, 100 mg |
| trypsin/EDTA | Cellgro | 25-053-CI | 0.25% Trypsin-EDTA 1X |
| DMEM | Life Technologies | 11965-092 | DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate |
| 10X PBS | Affymetrix | 75889 5 LT | PBS, 10X Solution, pH 7.4 |
| PFA (paraformaldehyde) | Affymetrix | 19943 1 LT | 4% in PBS |
| FBS | Sigma-Aldrich | F4135-500ML | Fetal Bovine Serum |
| EGM2-MV | Lonza | CC-3162 | EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. & Growth Factors |
| Triton X-100 | Sigma-Aldrich | T8787 | Triton X-100 |
| NaN3 | Sigma-Aldrich | S2002-25G | solubilized in water. 4% stock solution and working concentration is 0.4%. |
| mouse anti-human TM4SF1 antibody | Millipore | MAB3127 | Epitope is located in extracellular domain |
| mouse anti-human CD9 antibody | BD Bioscience | 555370 | Epitope is located in extracellular domain |
| Alexa Fluor 488 Donkey anti-mouse 2nd antibody | Life Technologies | A-21202 | Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) |
| Phalloidin | Chemicon | 90324 | Rhodamine-conjugated Phalloidin |
| anti-fade mounting media | Life Technologies | P-36931 | ProLong Gold Antifade Reagent with DAPI |
| Buffers and solutions | |||
| 70% ethanol | 17.5 ml of ethanol (200 proof) 7.5 ml of double deionized water |
||
| 10X PBS (make 200 ml) | |||
| 20 ml of 10X PBS | |||
| 180 ml of double deionized water | |||
| 20% Triton X-100 (make 20 ml) | |||
| 4 ml | |||
| 16 ml double deionized water | |||
| 4% NaN3 (make 25 ml) | |||
| 1 g of NaN3 | |||
| 25 ml of double deionized water | |||
| 50 ng/ml bovine collagen solution in PBS (make 5 ml) | |||
| Make stock soln. of 50 μg/ml (50 ml) | |||
| 830 μl of Collage I (3 mg) | |||
| 50 ml PBS | |||
| ICC Blocking Buffer (50 ml) | |||
| 49 ml PBS | |||
| 2% FBS (add 1 ml) | |||
| 0.04% NaN3 (add 100 μl of 4% NaN3) | |||
| ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml) | |||
| 50 ml of ICC Blocking Buffer | |||
| 25 μl of 20% Triton X-100 | |||
| Name of Cell | |||
| HUVEC | Lonza | C2517A | http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx |
| PC3 | ATCC | CRL-1435 | http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7 |
| Name of Equipment | |||
| cell culture hood | NuAIRE | Nu-425-600 | NU-425 (Series 60) BIOLOGICAL SAFETY CABINET |
| 37 °C, 5% CO2 cell culture incubator | CellStar | QWJ300DABA | Cellstar CO2 Water Jacketed Incubator |
| heating pad | K&H Manufacturing | 1020 | K&H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5×28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y) |
| centrifuge | Sorvall | T6000B | Sorvall T6000 (B) Benchtop Centrifuge |
| Hemacytometer | Sigma-Aldrich | Z359629 | Bright-Line Hemacytometer |
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