Physics Department E22, Technical University Munich, Garching, Germany.
Biophys J. 2011 Mar 16;100(6):1428-37. doi: 10.1016/j.bpj.2011.01.069.
We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15-30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width <v(1/2)>, ∼0.02 μm s(-1)) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10(-23) J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca(2+) by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.
我们通过细菌外套蛋白入侵（invasin）将磁镊与尖端耦合，从而研究了丝状伪足在力作用下的伸长情况。这种方法将力发生器与肌动蛋白束（通过肌球蛋白 X）耦合，从而阻碍了肌动蛋白加端的生长。我们施加了幅度在 20 到 600 pN 之间的单脉冲力（15-30 秒）和阶梯式力场景（幅度约为 50 pN；步宽为 30 秒）。在这两种情况下，响应都由快速粘弹性挠度和线性流动阶段组成。在关闭力后，挠度是可恢复的，这表明存在机械记忆。伸长速度呈指数分布（半宽 <v(1/2)>，约 0.02 μm s(-1))，并且没有随着力幅度的增加而系统增加。通过分析丝状伪足弯曲波动，我们估计了弯曲模量（0.4 × 10(-23) J m）和肌动蛋白丝的数量（~10）。通过 BAPTA 螯合细胞内 Ca(2+)会导致伸长幅度的强烈降低，而 latrunculin A 则导致弹性响应的丧失。我们将与力无关的速度归因于力诱导的肌动蛋白膜解耦，以及由 treadmilling 机制和弹性响应实现的可逆性，从而实现了肌动蛋白束的伸长。
