Generation of Directed Motion: How Random Steps in Cytoskeletal Systems can lead to Processive Movement

细胞内的运输方式多种多样：从浸泡在胞浆中的成分的纯扩散、随机运动，到分子马达的高度定向、确定性的移位。后者甚至可以连续执行多个步骤，这种能力被称为过程性。我们在这里要研究的是随机运动和高度进行性运动之间的广泛光谱。虽然大多数马达蛋白是严格结合在货物上的，但其中一些只是松散地结合在一起。例如，在细胞分裂期间，微管之间的相对滑动是由扩散锚定的运动蛋白马达促进的。在这一过程中产生了什么力量？这种类似于“降低”过程的扩散锚定有什么好处？另一方面，细胞骨架驱动的加工性也可以提高：蛋白质福尔明增强了聚合驱动的、热棘轮样的肌动蛋白细丝的向前运动。与没有成型的情况相比，聚合过程中的步长和作用力如何？而且，在一个很小的肌动蛋白束中，相当随机和真正非进行性的肌动蛋白细丝解聚能否产生足够的熵变化，使组装沿束轴收缩？利用应用于体外重组分析的单分子技术，我们将表征(I)由扩散锚定的kinesin-14马达驱动的微管-微管滑动，(Ii)在福尔明存在下肌动蛋白细丝的聚合，以及(Iii)细丝解聚引起的肌动蛋白束的收缩。我们认为这些过程是随机的、扩散的成分和定向的、确定的成分之间细胞内相互作用的原型系统。该项目的成果有望增加我们对生物系统如何利用不同的运动模式以及如何将这些模式进行有益的组合和相互转换的(生物)物理理解。

Intracellular transport comes in a wide variety: from the purely diffusive, random motion of components immersed in the cytosol to the highly directed, deterministic translocation of molecular motors. The latter may even perform multiple steps in a row, an ability referred to as processivity. It is the wide spectrum between random and highly processive motion that we will investigate here. While most motor proteins are rigidly bound to their cargo, some of them are only loosely attached. For example, during cell division the relative sliding of microtubules against each other is facilitated by diffusively anchored kinesin motors. What forces are generated during this process and what are the benefits of such diffusive anchorage, similar to a 'lowered' processivity? On the other hand, the processivity of cytoskeletal actuation can also be increased: the protein formin enhances the polymerization-driven, thermal-ratchet-like forward motion of actin filaments. How do step size and force during polymerization compare to the case without formin? And, can the rather random and truly non-processive depolymerization of actin filaments in a small actin bundle generate a sufficient entropic change to contract the assembly along the bundle axis? Using single-molecule techniques applied to reconstituted in vitro assays, we will characterize (i) the microtubule-microtubule sliding actuated by diffusively anchored kinesin-14 motors, (ii) the polymerization of actin filaments in the presence of formin, and (iii) the contraction of actin bundles induced by filament depolymerization. We regard these processes as prototypical systems for the intracellular interplay between random, diffusive elements and directed, deterministic constituents. The results of the project are expected to add to our (bio)physical understanding of how biological systems utilize different modes of movement as well as how these modes can be beneficially combined and interconverted.