Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin
Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin
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DOI：
10.1016/j.bpj.2009.03.046
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发表时间：
2009-06-17
影响因子：
3.4
通讯作者：
Janmey, Paul A.
中科院分区：
文献类型：
--
作者：
Byfield, Fitzroy J.;Wen, Qi;Janmey, Paul A.
来自许多组织的细胞类型通过主动重塑来响应基底硬度的变化。它们的细胞骨架来改变扩散面积或粘附强度，并且在某些情况下改变它们自身的硬度以匹配它们的基质。这些细胞对底物硬度的反应与底物诱导的许多蛋白质的状态、定位和量的变化有关，但在这些不同形式的机械反应中需要特定蛋白质的详细证据很少。这里我们使用微流体;技术来生产具有刚度梯度的凝胶，以显示细丝蛋白A在细胞对机械刺激的反应中的基本功能，并通过对比一对在该肌动蛋白交联蛋白的表达方面不同的人黑素瘤衍生细胞系的反应来解离细胞伸展和硬化。当M2黑色素瘤细胞仅通过胶原蛋白与基质连接时，细丝蛋白A无效的M2黑色素瘤细胞不改变其粘附面积以响应增加的基质硬度。受体，但通过纤连蛋白受体结合时正常改变粘附面积。相比之下，富含细丝蛋白A的A7细胞改变了两种基质上的粘附面积，并且对I型胶原包被的凝胶的反应比对纤连蛋白包被的凝胶更强烈。引人注目的是，A7细胞改变了它们的刚度，如通过原子力显微镜测量的，以立即匹配梯度上与它们相邻的基底的弹性模量。相反，M2细胞在所有基质上保持恒定的刚度，其与A7细胞在所检查的最软凝胶上的刚度一样低（11000 Pa）。在相同梯度基底上的细胞铺展和细胞硬化的比较表明，细胞铺展与硬化是解耦的。在饱和的胶原蛋白和纤连蛋白浓度下，与A7细胞相比，M2细胞的粘附减少到大约等于粘附面积差异的程度。细丝蛋白A似乎是必不可少的胶原蛋白上的细胞硬化，但不是细胞的纤连蛋白上的蔓延。这些结果对细胞突起和粘附的不同模型具有影响，并确定细丝蛋白A在改变细胞刚度中的关键作用，该作用不能通过体内其他肌动蛋白交联剂来补偿。
Cell types from many tissues respond to changes in substrate stiffness by actively remodeling. their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous, proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics; techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen. receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately, adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (11000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.