Pallavicini Carla, Levi Valeria, Wetzler Diana E, Angiolini Juan F, Benseñor Lorena, Despósito Marcelo A, Bruno Luciana
Departamento de Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.
Biophys J. 2014 Jun 17;106(12):2625-35. doi: 10.1016/j.bpj.2014.04.046.
The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.
细胞骨架参与众多细胞过程，如迁移、分裂和收缩，并为分子马达驱动的运输提供轨道。因此，量化细胞骨架细丝的力学行为对于更好地理解细胞力学和组织非常重要。已经证明，微管的相关力学性质可以从其运动和形状波动的分析中提取。然而，在活细胞中追踪单个细丝极其复杂，例如，由于背景高且不均匀。我们引入了一种新的追踪算法，该算法能够在体外条件下以约9纳米的精度恢复荧光微管的坐标。为了说明该算法的潜在应用，我们研究了活细胞中荧光微管的曲率分布。通过对微管形状进行傅里叶分析，我们发现曲率遵循先前报道的类似热分布，有效持续长度约为20微米，该值明显小于在体外测量的值。我们还验证了微管相关蛋白XTP或肌动蛋白网络的解聚不会影响该值；然而，中间丝的破坏会降低持续长度。此外，我们恢复了肌动蛋白或中间丝缺失细胞中微管片段的轨迹，并观察到它们相对于未处理细胞的运动显著增加，表明这些细丝有助于微管网络的整体组织。此外，对未处理细胞中微管片段轨迹的分析表明，这些细丝在皮层中相对于核周区域呈现出较慢但更具方向性的运动，这表明该追踪程序将允许绘制细胞中微管的动态组织图。
