Jiang Guoying, Huang Angela H, Cai Yunfei, Tanase Monica, Sheetz Michael P
Department of Biological Sciences, Columbia University, New York, New York 11027, USA.
Biophys J. 2006 Mar 1;90(5):1804-9. doi: 10.1529/biophysj.105.072462. Epub 2005 Dec 9.
Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.
细胞需要最佳的底物硬度来实现正常功能和分化。然而，感知基质硬度和趋硬性的机制尚不清楚。在这里，我们发现对照细胞系、Shp2基因敲除细胞系、整合素β1基因敲除细胞系和踝蛋白1基因敲除细胞系在纤连蛋白（FN）包被的刚性聚丙烯酰胺表面上的铺展面积均比在柔软表面上大三倍。相比之下，RPTPα基因敲除细胞在FN表面上的铺展面积与表面硬度无关，但在刚性IV型胶原包被的表面上的铺展面积比在柔软表面上大3倍。先前已证明RPTPα和αvβ3整合素共定位于前缘，并且αvβ3抗体可阻断FN硬度感知。当用刚性激光阱在前缘捕获FN珠时，与细胞骨架形成的键比用柔软激光阱捕获时更强；而在前缘后方以及在RPTPα基因敲除细胞中，用刚性和柔软激光阱形成的键都较弱。根据陷阱的硬度，我们计算出在1秒内产生10皮牛的力足以激活硬度反应。我们认为，前缘处的RPTPα和αvβ3可能是通过前缘处的Src家族激酶（SFK）激活和下游信号传导来感知FN基质硬度的关键元件。
