Actin/myosin function in intracellular vesicle transport processes

膜定向运输是真核细胞组织和极化的基础。沿着微管和肌动蛋白细丝轨迹的联合运输允许分子在空间和时间上的特定输送。Rab11 GTPase是胞外和循环途径细胞内转运过程的主要调节者，因此在细胞表面蛋白表达的调控中起着关键作用。作为一个分子开关，Rab11在囊泡和内膜上构建不同的马达蛋白复合体，使动蛋白和动力蛋白能够介导沿微管的快速和远程运输，以及肌球蛋白V肌动蛋白马达蛋白沿肌动蛋白细丝的较慢和局部运输。我们最近的发现表明，Spir肌动蛋白核苷和肌球蛋白V与Rab11在囊泡膜上组装成一个三方复合体，表明在Rab11囊泡上肌动蛋白细丝轨迹和肌球蛋白力的产生是协调的。这使得肌动蛋白/MyoV驱动的运动在远离皮质肌动蛋白网络的细胞区域中进行，例如在卵母细胞的细胞质中，很可能在体细胞的中央区域。基于活体细胞成像数据显示频繁的Spir/Rab11囊泡形成纳米管，我们提出了一个模型，在该模型中，Spir和MyoV的合作为囊泡的形态动力学提供了力量，从而促进了囊泡附着到微管上进行远程运输。新的基因组编辑技术应该被用来特异性地损害或改变小鼠成纤维细胞中的Spir和MyoV功能。荧光蛋白标记的Spir、MyoV和Rab11应在其内源性启动子的调控下表达。现代荧光活细胞成像技术将使我们能够监测Rab11小泡，并将形态动力学和运动性与Spir和MyoV功能相关联。本文提出的研究将提供关于细胞内小泡运输过程中肌动蛋白/肌球蛋白力功能的基本知识，从而为后续依赖这些功能的细胞生物学过程的机制分析奠定基础，如黏附分子和生长因子受体的表面表达。

Directional membrane transport is the basis of eukaryotic cell organisation and polarisation. A combined transport along microtubule and actin filament tracks allows a specified delivery of molecules in space and time. The Rab11 GTPase is a master regulator of intracellular transport processes of the exocytic and recycling pathways and thereby a key function in the regulation of cell surface protein expression. Acting as a molecular switch, Rab11 builds distinct motor protein complexes at vesicle and endosomal membranes, which enable kinesin and dynein mediated fast and long-range transport along microtubules, as well as slower and local transport by myosin V actin motor proteins along actin filaments.Our recent discovery that Spir actin nucleators and myosin V assemble with Rab11 into a tripartite complex at vesicle membranes indicates a coordinated nucleation of actin filament tracks and myosin force generation on a subset of Rab11 vesicles. This enables actin/MyoV-driven motility in regions of the cell distal from the cortical actin network, such as in the oocyte cytoplasm and most likely in the central region of somatic cells. Based on live cell imaging data showing frequent nanotube formations of Spir/Rab11 vesicles, we propose a model in which the Spir and MyoV cooperation provides forces for vesicular morphological dynamics, which facilitates the attachment of vesicles to microtubule tracks for long-range transport.Novel genome editing technologies should be employed to specifically impair or modify Spir and MyoV functions in mouse fibroblast cells. Fluorescent protein tagged Spir, MyoV and Rab11 should be expressed under the control of their endogenous promoters. Modern fluorescence live cell imaging technologies will then enable us to monitor Rab11 vesicles and correlate morphological dynamics and motility to Spir and MyoV functions.The study proposed here should provide basic knowledge on the yet poorly understood functions of actin/myosin forces in intracellular vesicle transport processes and should thereby be elementary for subsequent mechanistic analysis of cell biological processes depending on these functions, such as the surface expression of adhesion molecules and growth factor receptors.