富德瑞迪蒂菲洛波迪亚成分的纯化
Summary
在此协议中,我们介绍了一种利用树突状纤维蛋白之间特定且强烈的亲和力,从培养的海马神经元上的方皮杯状突起结构中纯化树突状纤维素的富足部分的方法。粘附分子,TLCN,和细胞外基质分子,体外内丁。
Abstract
丹德林丝洛波迪亚是薄和长突起基于作用素灯丝,他们延伸和缩回,仿佛寻找目标斧突。当树突状纤维化与目标斧突建立接触时,它们开始成熟成脊柱,导致突触的形成。端脑素 (TLCN) 在树突状纤维化中大量局部化,并逐渐被排除在脊柱之外。在培养的海马神经元中TLCN的过度表达诱导树突状的纤维化形成。我们表明,端脑蛋白与细胞外基质分子,体外核酸强结合。Vitronectin涂层微珠诱导神经元树突形成方位细胞杯。在方细胞杯中,TLCN、TLCN结合蛋白,如磷酸化Ezrin/Radixin/Moesin(磷-ERM)和F-actin被积累,这表明方位细胞杯的成分与树突状西多迪亚的成分相似。因此,我们开发了一种净化方丹花杯的方法,而不是树突状的纤维化。磁聚苯乙烯珠被涂覆体内蛋白,在海马神经元的培养基中大量存在,诱导神经元树突形成方位体。孵育24小时后,用洗涤剂温和溶解咽喉杯,并用磁铁分离器收集。洗涤珠子后,通过银染色和西方印迹对结合蛋白进行洗脱和分析。在结合部分中,TLCN和actin大量存在。此外,从该分数中识别的许多蛋白质被局部化为树突状纤维化;因此,我们命名结合分数作为树突状的菲洛波迪亚丰富的分数。本文介绍了树突状纤维素富分馏的纯化方法。
Introduction
丹德炎纤维素被认为是脊柱的前体。在树突状纤维中的Actin细丝调节其延伸和缩回1,2,3。与斧子接触后,选定的树突状纤维开始成熟到脊柱,并形成突触形成4,5。脊柱的成分是由对鼻后密度分数6、7的综合分析确定的,而树突状纤维素的成分在很大程度上仍不为人所知。已经表明,端脑素(TLCN),ERM,SynGAP,Ras,PI3激酶,阿克特,mTOR,马球状激酶2,CaMKII,Syndecan-2,帕拉lemin-1,ARF6和EphB调节树突状纤维素形成5,8,9 ,10,11,虽然尚未开发一种方法,用于综合分析存在于树突状的纤维素中的分子。
TLCN(ICAM-5)是由最神经部分的尖刺神经元,即端脑12中专门表达的。TLCN在其细胞外区域有9个类似Ig的域,一个跨膜区域,一个细胞质尾13。TLCN与其细胞外区域的体外核酸素(VN)和LFA-1一元结结合,在其跨膜区域与前塞尼林结合,在其细胞质区5、8、14、15中与磷-ERM和β-actinin结合 ,16.TLCN通过磷-ERM结合到脊柱和树突状轴8、16的树突状纤维素和β-actinin的尖端。
研究表明,TLCN的过度表达增强了树突状纤维化的形成,并诱导脊柱回归到纤维化10。与TLCN细胞质区结合的ezrin的构成活性形式,增强树突状纤维化形成8。因此,TLCN通过行动素结合蛋白调节树突状纤维化形成。埃塞伦斯等人证明,微珠诱导TLCN在培养神经元上积累17。研究表明,在VN涂层微珠周围的神经元树突上形成方状的杯子结构以TLCN依赖性的方式形成15。树突状纤维素的成分与咽喉杯的成分相似。收集树突状纤维素是很困难的,但使用磁性微珠收集方位细胞杯相对容易。因此,我们开发了一种净化方丹石杯的方法,而不是树突状的纤维化18。在这里,我们描述了树突状富足部分的纯化方法。
Protocol
Representative Results
Discussion
利用细胞粘附分子TLCN与细胞外基质蛋白体外核酸之间的亲和力,开发了一种树突状纤维素富度分体的纯化方法。与PSD分数相比,有可能从富含树突子的丝虫片段中识别作用于未成熟突触的突触蛋白。因此,富含树突的纤维素的成分与PSD分数的成分相差74%。与PSD分数不同,我们使用培养的海马神经元主动形成噬菌体杯,细胞需要存活。为了形成方位细胞杯,我们使用了TLCN和体外核子细胞之间的相互作用。TL…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢小冈和松野幸男为海马神经元的低密度培养,Masayoshi Mishina为TLCN缺乏小鼠,三井三子和MomokoShiozaki的技术援助,以及Yoshihara实验室的成员进行有益的讨论.这项工作得到了JSPS KAKENHI格兰特Nos的支持。JP20700307、JP22700354 和 JP24500392 和 MEXT KAKENHI 授予号。JP23123525 到 YF 和 JP20022046、JP18H04683 和 JP18H05146 到 YY。
Materials
| 1 M HEPES | Gibco | 15630-080 | |
| 1.7 ml Low Binding MCT | Sorenson BioScience | 39640T | |
| 200 mM L-Glutamine | Gibco | 2530149 | |
| 35-mm plastic cell culture dishes | Corning | 430165 | |
| Anti-actin | Sigma-Aldrich | A-5060 | |
| Anti-alpha-Actinin | Sigma-Aldrich | A-5044 | |
| Anti-alpha-tubulin | Sigma-Aldrich | T-9026 | |
| Anti-Ezrin | Sigma-Aldrich | clone3C12, SAB4200806 | |
| Anti-Galphaq | Santacruz | sc-393 | |
| Anti-MAP2 | Chemicon | clone AP20, MAB3418 | |
| Anti-Moesin | Sigma-Aldrich | clone 38/87, M7060 | |
| Anti-PLCbeta1 | Santacuz | sc-5291 | |
| Anti-PSD95 | MA2 | ABR | |
| Anti-Spectrin beta | Chemicon | MAB1622 | |
| B27 | Gibco | 0080085SA | |
| BCA protein assay kit | Thermo | 23227 | |
| Bromophenol blue | Merck | 1.08122.0005 | |
| calcium chrolide, hydrous | Wako | 038-19735 | |
| Cell scraper | Falcon | 353085 | |
| Cell strainer | Falcon | 352350 | |
| Choline chloride | Sigma-Aldrich | C7527 | |
| Complete EDTA free protease inhibitor cocktail | Roche | 11873580001 | |
| Cytosine beta-D-arabinofuranoside | Sigma-Aldrich | C-6645 | |
| DNase-I | Sigma-Aldrich | DN-25 | |
| D-Pantothenic acid hemicalcium salt | Sigma-Aldrich | P5155 | |
| DynaMag-2 Magnet | Thermo | 12321D | |
| ECL Prime Western Blotting Detection Reagent | GE | RPN2232 | |
| e-PAGEL 5-20% SDS-PAGE gradient gel | ATTO | E-T520L | |
| Folic acid | Sigma-Aldrich | F8758 | |
| HBSS | Gibco | 14175095 | |
| HRP-conjugated anti-rabbit IgG | Jackson ImmunoResearch | 111-035-144 | |
| i-Inositol | Sigma-Aldrich | I7508 | |
| LAS-1000 mini | Fuji Film | LAS-1000 mini | For detection of luminescence from WB membrane |
| Magnetic polystyrene microbeads | Sperotech | PM-20-10 | |
| MEM amino acid solution | Gibco | 11130-051 | 30 mM L-Arginine hydrochloride, 5 mM L-Cystine, 10 mM L-Histidine hydrochloride-H2O, 20 mM L-Isoleucine, 20 mM L-Leucine, 19.8 mM L-Lysine hydrochloride, 5.1 mM L-Methionine, 10 mM L-Phenylalanine, 20 mM L-Threonine, 2.5 mM L-Tryptophan, 10 mM L-Tyrosine, and 20 mM L-Valine |
| Mini-slab size electrophoresis system | ATTO | AE-6530 | |
| Niacinamide | Sigma-Aldrich | N0636 | |
| Penicilin / Streptomycin | Gibco | 15070063 | |
| PhosSTOP phosphatase inhibitor cocktail | Roche | 4906845001 | |
| Poly-L-lysine hydrobromide | Nacali | 28360-14 | |
| Pyridoxal HCl | Sigma-Aldrich | P6155 | |
| Riboflavin | Sigma-Aldrich | R9504 | |
| Silver Stain 2 Kit wako | Wako | 291-5031 | |
| Thiamine HCl | Sigma-Aldrich | T1270 | |
| Trans-Blot SD Semi-Dry Transfer Cell | Bio-rad | 1703940JA | |
| Ultra pure water | MilliQ | For production of ultra pure water |
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