Using self-assembled monolayers to model cell adhesion to the 9th and 10th type III domains of fibronectin.
Using self-assembled monolayers to model cell adhesion to the 9th and 10th type III domains of fibronectin.
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DOI：
10.1021/la901528c
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发表时间：
2009-12-15
期刊：
影响因子：
--
通讯作者：
Mrksich M
中科院分区：
文献类型：
--
作者：
Eisenberg JL;Piper JL;Mrksich M
大多数哺乳动物细胞必须粘附于细胞外基质（ECM）以维持正常的生长和发育。纤连蛋白是一种主要的 ECM 蛋白，通过其 Arg-Gly-Asp (RGD) 和 Pro-His-Ser-Arg-Asn (PHSRN) 肽结合位点与整合素细胞受体结合。为了研究这些基序在细胞粘附中发挥的作用，来自第 9 个（包含 PHSRN）和第 10 个（包含 RGD）III 型纤连蛋白结构域的蛋白质被设计为与角质酶（一种丝氨酸酯酶，与磷酸配体形成位点特异性共价加合物）符合框架。在三（乙二醇）基团惰性背景下呈现膦酸配体的自组装单层（SAM）被用作固定角质酶-纤连蛋白融合蛋白的模型底物。小仓鼠肾细胞可有效附着在所有蛋白质表面，但仅在含有 RGD 肽的蛋白质单层上有效扩散。与 PHSRN 呈递表面上的细胞相比，含有 RGD 的蛋白质表面上的细胞还表现出明确的粘着斑和有组织的细胞骨架结构。与呈现较高蛋白质密度的 SAM 上单独的 RGD 相比，细胞附着和扩散不受 PHSRN 存在的影响，但当 RGD 以低蛋白质密度呈现时，PHSRN 支持细胞附着效率的提高。用可溶性 RGD 或 PHSRN 肽处理悬浮细胞表明，这两种肽都能抑制 FN10 表面的附着。这些结果支持这样一个模型：PHSRN 和 RGD 竞争性地与整合素结合，而不是两点协同相互作用，并且 PHSRN 的存在有助于增加基质上配体的密度，从而提高细胞在附着过程中的粘附概率。
Most mammalian cells must adhere to the extracellular matrix (ECM) to maintain proper growth and development. Fibronectin is a predominant ECM protein that engages integrin cell receptors through its Arg-Gly-Asp (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) peptide binding sites. To study the roles these motifs play in cell adhesion, proteins derived from the 9th (containing PHSRN) and 10th (containing RGD) type III fibronectin domains were engineered to be in frame with cutinase, a serine esterase that forms a site-specific, covalent adduct with phosphonate ligands. Self-assembled monolayers (SAMs) that present phosphonate ligands against an inert background of tri(ethylene glycol) groups were used as model substrates to immobilize the cutinase-fibronectin fusion proteins. Baby hamster kidney cells attached efficiently to all protein surfaces, but only spread efficiently on protein monolayers containing the RGD peptide. Cells on RGD-containing protein surfaces also displayed defined focal adhesions and organized cytoskeletal structures compared to cells on PHSRN-presenting surfaces. Cell attachment and spreading were shown to be unaffected by the presence of PHSRN when compared to RGD alone on SAMs presenting higher densities of protein, but PHSRN supported an increased efficiency in cell attachment when presented at low protein densities with RGD. Treatment of suspended cells with soluble RGD or PHSRN peptides revealed that both peptides were able to inhibit the attachment of FN10 surfaces. These results support a model wherein PHSRN and RGD bind competitively to integrins―rather than a two-point synergistic interaction―and the presence of PHSRN serves to increase the density of ligand on the substrate and therefore enhance the sticking probability of cells during attachment.
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影响因子：
3.7
作者：
DANILOV, YN;JULIANO, RL
通讯作者：
JULIANO, RL
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影响因子：
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作者：
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DOI：
10.1073/pnas.072685299
发表时间：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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