Covalent and density-controlled surface immobilization of E-cadherin for adhesion force spectroscopy.
Covalent and density-controlled surface immobilization of E-cadherin for adhesion force spectroscopy.
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DOI：
10.1371/journal.pone.0093123
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Franz CM
中科院分区：
文献类型：
--
作者：
Fichtner D;Lorenz B;Engin S;Deichmann C;Oelkers M;Janshoff A;Menke A;Wedlich D;Franz CM
E-钙粘蛋白是一种关键的细胞间粘附分子，但受体密度的影响以及单个钙粘蛋白胞外域在促进细胞粘附方面的精确贡献尚不完全清楚。研究这些机制将受益于在确定的表面密度下携带不同钙粘蛋白胞外域的人工粘附基质。因此，我们开发了基于 SNAP 标签技术的定量 E-钙粘蛋白表面固定方案。与 SNAP 标签融合的 E-钙粘蛋白的胞外 (EC) 片段与携带苄基鸟嘌呤 (BG) 头基的硫醇自组装单层 (SAM) 共价结合。然后使用细胞铺展测定以及单细胞（SCSF）和单分子（SMSF）力谱评估不同E-钙粘蛋白表面的粘附功能。我们证明，仅包含第一和第二最外层 EC 结构域 (E1-2) 的 E-钙粘蛋白构建体不足以介导细胞粘附，并且仅产生较低的单钙粘蛋白-钙粘蛋白粘附力。相比之下，包含所有五个 EC 结构域 (E1-5) 的构建体可有效促进细胞扩散并产生强大的单个钙粘蛋白和细胞粘附力。通过改变 SAM 内 BG 头基的浓度，我们确定了 5-11 nm 的横向距离，以实现最佳的 E-钙粘蛋白功能。整合 SCMS 和 SMSF 实验的结果进一步表明，E-钙粘蛋白粘附接触的溶解涉及单个钙粘蛋白受体的连续解离，而不是较大钙粘蛋白受体簇的突然破裂。因此，我们的共价、定向和密度控制的 E-钙粘蛋白固定方法提供了一个新颖且多功能的平台，用于在确定的实验条件下研究钙粘蛋白介导的细胞粘附的分子机制。
E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5–11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.
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DOI：
10.1083/jcb.201110076
发表时间：
2012-08-20
期刊：
The Journal of cell biology
影响因子：
--
作者：
Kraft B;Berger CD;Wallkamm V;Steinbeisser H;Wedlich D
通讯作者：
Wedlich D
影响因子：
19
作者：
Brasch J;Harrison OJ;Honig B;Shapiro L
通讯作者：
Shapiro L
影响因子：
4.6
作者：
del Valle, Ignacio;Rudloff, Stefan;Kemler, Rolf
通讯作者：
Kemler, Rolf
DOI：
10.1073/pnas.070052697
发表时间：
2000-04-11
影响因子：
11.1
作者：
Baumgartner, W;Hinterdorfer, P;Drenckhahn, D
通讯作者：
Drenckhahn, D
影响因子：
1.6
作者：
HUTTER, JL;BECHHOEFER, J
通讯作者：
BECHHOEFER, J