Institut de Ciències Fotòniques (ICFO), 08860 Castelledefels (Barcelona), Spain.
Proc Natl Acad Sci U S A. 2013 Feb 26;110(9):3375-80. doi: 10.1073/pnas.1219206110. Epub 2013 Feb 11.
Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubule-microtubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than ∼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network.
细胞内运输在维持极化细胞的组织中起着至关重要的作用。在长距离运输过程中，马达蛋白将货物与微管连接并沿微管移动，以将其运送到适当的功能位置。为了到达目的地，货物结合的马达必须克服前进运动的障碍，例如微管之间的交点。能够可视化马达如何克服这些障碍，可以提供有关导致有效运输的机制的重要信息。在这里，我们首先开发了一种基于单粒子跟踪和超分辨率显微镜的全光学相关成像方法，以高时空分辨率将货物的运输轨迹映射到单个微管上。然后，我们使用该方法研究了溶酶体在微管-微管交点处的行为。我们的结果表明，当相交微管的分离距离小于约 100nm 时，交点会造成显著的阻碍，导致运输长时间停顿。然而，马达通常可以通过切换到相交的微管或以高保真度通过交点来克服这些障碍物。有趣的是，在交点之后，存在保持运动极性（正向或逆向）的大趋势，这表明对马达活性的高度调节以维持在给定方向上的运输。这些结果深入了解了细胞骨架几何形状对货物运输的影响，并对货物结合的马达用于通过复杂细胞骨架网络设置的障碍物进行操纵的机制具有重要意义。
