Schmidt C E, Horwitz A F, Lauffenburger D A, Sheetz M P
Department of Chemical Engineering, University of Illinois, Urbana 61801.
J Cell Biol. 1993 Nov;123(4):977-91. doi: 10.1083/jcb.123.4.977.
We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin-cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body.
我们利用激光光镊和纳米级运动分析技术，研究了迁移的成纤维细胞背表面上的β1整合素（一种细胞-底物粘附分子）的细胞骨架关联和表面动力学。使用单光束光学梯度阱（激光镊子）来限制与抗β1整合素单克隆抗体偶联的聚苯乙烯珠，并将它们放置在细胞外的所需位置。该技术用于证明迁移细胞中整合素-细胞骨架相互作用的空间差异。我们发现，与收缩部分相比，运动细胞的片状伪足上珠子的稳定附着以及随后的向后流动明显增加。与细胞片状伪足处整合素的增强连接互补的是，运动细胞后部的膜比前部更易变形，而非运动细胞在膜结构上没有表现出这种不对称性。通过监测涂有抗β1整合素单克隆抗体的胶体金颗粒的位移，利用视频显微镜和纳米精度跟踪程序研究了迁移细胞片状伪足上整合素的表面动力学。小金聚集体优先快速运输到片状伪足的前沿，在那里它们恢复沿边缘受限的扩散。这种快速运输的特征是短暂的定向运动（“跳跃”），瞬时速度为37±15微米/分钟（标准差），中间间隔扩散期。相比之下，较大的金颗粒聚集体和大乳胶珠以类似于其他帽化事件以及这些细胞迁移所报道的速率进行缓慢、稳定的向后运动（0.85±0.44微米/分钟）（标准差）。含有突变β1整合素的细胞系用于表明细胞质结构域对于整合素与潜在细胞骨架网络附着的不对称性至关重要，并且对于快速、间歇性运输也是必需的。然而，细胞后部增强的膜可变形性并不需要整合素-细胞骨架相互作用。我们还证明了整合素的翻译后修饰可能在这些现象中起作用。这些结果提出了一个动态整合素介导的粘附相互作用在细胞迁移中作用的方案。整合素优先运输到细胞前端，在那里它们形成新生粘附。这些粘附结构尺寸增大并与对其施加向后力的细胞骨架相关联。背侧聚集体更向后移动，而腹侧的聚集体则保持固定在底物上，从而使细胞体向前移动。细胞后部的脱离至少通过两种方式发生：（a）整合素-细胞骨架相互作用减弱，可能由连接蛋白的局部修饰介导，这导致细胞-底物相互作用减弱；（b）整合素和高度可变形的膜从细胞体撕裂。
