Membrane Biophysics of African Trypanosomes

昏睡病对撒哈拉以南非洲的人类和牲畜构成严重威胁。它是由锥虫引起的，锥虫是一种单细胞鞭毛虫，是膜生物物理领域研究的优秀模式生物。在宿主的血液中，锥虫的质膜被一层致密的、几乎均匀的变异表面糖蛋白（VSGs）覆盖。这种高度动态的表面涂层至关重要，因为它可以保护寄生虫免受宿主免疫系统的侵害。由于其蛋白质密度，VSG涂层构成了一个有希望的框架来研究大分子拥挤，并允许与人工模型膜进行比较测量。弥散的VSG蛋白的胞内和胞外作用局限于鞭毛囊。这种情况让人想起所谓的二维窄逃逸问题（NEP）。NEP是生物学和生物物理学中的一个常见问题。它处理的布朗粒子被限制在一个给定的有反射边界的区域，只有一个小的开口，在那里粒子被吸收。应用现有的NEP解析解计算VSG找到鞭毛袋所需的时间与实验结果有明显的差异。我们将用两种不同的方式来解决这个问题。首先，利用单分子荧光显微镜测量活体布氏体内外膜的VSG动态，寻找非布朗组分。我们的目标是确定物理效应的影响，如曲率辅助分选，而不是活跃的细胞依赖的贡献。选择具有良好特征的布氏体作为模式生物使我们能够直接测试活跃参与者的贡献，例如分子马达，在敲除实验中。第二，通过实验挑战NE理论。为了测试分析解的适用性，我们计划在微图案模型膜中进行系统研究，允许我们改变几何参数，并在宽相空间中测试理论模型的有效性。此外，我们的目标是通过敲低相关结构蛋白来操纵体内的几何形状。这个项目的目标是实现对锥虫生理学和基本新经济政策的更深层次的理解。最终，了解VSG动力学背后的物理参数可能是开发替代锥虫剂的基础，这些替代锥虫剂旨在改变这些参数，从而不易诱导抗性。

Sleeping sickness poses a serious threat to humans and livestock in sub-Saharian Africa. It is caused by the trypanosome parasite, a unicellular flagellate and excellent model organism for research in the field ofmembrane biophysics.In the bloodstream of the host the trypanosome plasma membrane is covered with a dense, almost uniform coat of variant surface glycoproteins (VSGs). This highly dynamic surface coat is of vital importance because it protects the parasite from the immune system of the host. Due to its protein density the VSG coat constitutes a promising framework to study macromolecular crowding as well as allows comparative measurements with artificial model membranes. Endo- and exocytosis of diffusing VSG proteins are restricted to the flagellar pocket. This scenario is reminiscent of the so-called narrow escape problem (NEP) in two dimensions. The NEP is a common problem in biology and biophysics. It deals with Brownian particles confined to a given domain with reflecting borders and only a small opening where the particles are absorbed. Applying the currently available analytical solution of the NEP to calculate the time a VSG needs to find the flagellar pocket yields a clear discrepancy with experimental results. We will address the problem in two different ways. First, by measuring VSG dynamics in the external and internal membranes of T. brucei in vivo with single-molecule fluorescence microscopy and looking for non-Brownian components. We aim to identify the influence of physical effects like curvature-assisted sorting in contrast to active cell-dependent contributions. Choosing the well-characterized T. brucei as a model organism allows us to directly test contributions of active players, e.g. molecular motors, in knock-down experiments.Second, by challenging the theory of NE experimentally. In order to test the applicability of the analytical solution, we plan a systematic study in micro-patterned model membranes that allows us to vary geometric parameters and test the validity of the theoretical model in a wide phase space. Moreover, we aim to manipulate the in vivo geometry by knockdown of relevant structural proteins. It is the objective of this project to achieve both a deeper understanding of trypanosome physiology and the fundamental NEP. Ultimately, knowledge of the physical parameters underlying VSG dynamics could be the basis for the development of alternative trypanocide agents that aim to change these parameters and are thus not susceptible to induce resistance.