Schmidt C E, Chen T, Lauffenburger D A
Department of Chemical Engineering, University of Illinois, Urbana 61801.
Biophys J. 1994 Jul;67(1):461-74. doi: 10.1016/S0006-3495(94)80502-8.
Cell migration is a dynamic phenomenon requiring a physical interaction between the internal cell motile machinery and the external substratum in which adhesion receptors, such as integrins, serve as the transmembrane link. To analyze quantitatively this interaction, we apply a modified Brownian dynamics algorithm to simulate cytoskeleton-mediated transport of integrin on the dorsal surfaces of migrating fibroblasts. Previously, we experimentally demonstrated that integrin is transported in an intermittent fashion, with directed excursions interspersed by diffusive periods, preferentially toward the cell edge where the integrin is likely used in the formation of nascent adhesions. Integrins containing mutations in the cytoskeleton-binding region of the cytoplasmic domain display statistically different degrees of directed transport, indicating that this phenomenon is dependent on cytoskeletal associations. In the present work, we develop a computer algorithm generating simulated integrin transport trajectories, given estimates for the rate constants defining coupling (kc) and uncoupling (ku) of integrin with cytoskeletal components. Other parameters supplied to the program, the diffusion coefficient (D) for integrin in the membrane and the instantaneous velocity (vi) of the integrin/cytoskeleton complex, have been measured independently in our experimental system. By comparing the simulated trajectories with those obtained experimentally, we are able to estimate the coupling and uncoupling rate constants for the interaction of integrin with cytoskeletal elements in vivo. We find that integrin couples with cytoskeletal elements at a rate approximately 10 times slower than its rate of uncoupling (kc = 0.3 s-1, ku = 3 s-1). Comparison of these rate constants with an equivalent rate constant for diffusion, k+ = 0.4 s-1, indicates that the coupling interaction is likely a diffusion-limited process, as is typically expected for membrane processes. We further show by calculation that directed transport is necessary for integrin to traverse the length of an extending lamellipod to its leading edge; diffusion alone is not sufficiently fast to supply adhesion receptors to points of new cell/substratum contact.
细胞迁移是一种动态现象，需要细胞内部运动机制与外部基质之间进行物理相互作用，其中诸如整合素等黏附受体充当跨膜连接。为了定量分析这种相互作用，我们应用一种改进的布朗动力学算法来模拟细胞骨架介导的整合素在迁移成纤维细胞背表面上的运输。此前，我们通过实验证明整合素以间歇性方式运输，定向偏移穿插着扩散期，优先朝着细胞边缘运输，在那里整合素可能用于新生黏附的形成。在细胞质结构域的细胞骨架结合区域含有突变的整合素显示出统计学上不同程度的定向运输，表明这种现象依赖于细胞骨架关联。在本工作中，给定定义整合素与细胞骨架成分耦合（kc）和解耦（ku）的速率常数估计值，我们开发了一种生成模拟整合素运输轨迹的计算机算法。提供给该程序的其他参数，即整合素在膜中的扩散系数（D）和整合素/细胞骨架复合物的瞬时速度（vi），已在我们的实验系统中独立测量。通过将模拟轨迹与实验获得的轨迹进行比较，我们能够估计整合素在体内与细胞骨架元件相互作用的耦合和解耦速率常数。我们发现整合素与细胞骨架元件耦合的速率比其解耦速率慢约10倍（kc = 0.3 s-1，ku = 3 s-1）。将这些速率常数与扩散的等效速率常数k+ = 0.4 s-1进行比较表明，耦合相互作用可能是一个扩散限制过程，这是膜过程通常所预期的。我们通过计算进一步表明，定向运输对于整合素穿越延伸的片状伪足长度到达其前沿是必要的；仅靠扩散速度不够快，无法将黏附受体供应到新的细胞/基质接触点。
