Ubiquitin-dependent and -independent functions of Myosin VI – Characterization of a molecular motor protein in DNA-replication and -repair and immune signaling

非常规肌球蛋白是基于肌动蛋白的分子运动蛋白，负责细胞内囊泡运输，结构维持和力产生过程。肌凝蛋白VI代表了肌凝蛋白超家族的一个独特成员，因为它的运动方向与迄今为止分析的所有其他肌凝蛋白相反。它参与多种信号通路（如内吞作用、分泌、自噬等），其失调常与人类疾病相关。在前列腺癌和卵巢癌中，肌球蛋白VI属于上调最多的基因。除了细胞质定位外，肌凝蛋白VI也被发现存在于细胞核中，尽管其核功能的特征不明显。我在Simona Polo博士实验室的第一个博士后期间，我们在Myosin VI （MIU和MyUb）的尾域内发现了两个泛素结合域（UBDs）。在这两个UBDs之间插入一个选择性剪接元件可导致至少三种不同Myosin VI亚型的形成。我们发现含有这种剪接插入的Myosin VI亚型主要与内吞机制的组分相互作用，而没有插入的Myosin VI亚型与许多核相互作用物结合。有趣的是，癌细胞选择性地表达短异构体，表明Myosin VI的主要核功能与肿瘤发生有关。在Helle Ulrich教授的实验室（与Petra Beli合作），我证实并明确了Myosin VI的短亚型与核蛋白的相互作用，这些核蛋白参与了基本的基因组维持途径，如DNA修复、DNA复制和免疫信号传导。此外，通过细胞培养实验，我能够证明肌凝蛋白VI在上述途径中的功能重要性，其中我展示了DNA依赖的肌凝蛋白VI在细胞核丝状结构上的定位。该项目的目的是研究肌凝蛋白VI在细胞核和细胞质中的功能，重点是DNA复制和修复以及免疫信号传导。表征泛素依赖的过程应该揭示肌球蛋白VI调节途径的分子机制。我们期望对细胞骨架如何与染色质相互作用及其对核过程动力学的影响有新的见解。本项目所获得的结果将为了解前列腺癌和卵巢癌发生过程中的病理事件提供基础知识，从而有助于确定癌症治疗的新分子靶点。

Unconventional Myosins are actin-based molecular motor proteins responsible for intracellular vesicle transport, structural maintenance and force-generating processes. Myosin VI represents a unique member of the myosin superfamily because it moves in a direction opposite to all other myosins analysed so far. It is involved in various signalling pathways (e.g. endocytosis, secretion, autophagy, etc.) where its deregulation is often correlated with human diseases. In prostate and ovarian carcinoma, myosin VI belongs to the most heavily upregulated genes. Next to its cytoplasmic localization, Myosin VI was also found to be present in the nucleus, although its nuclear functions are poorly characterised.During my first postdoc in the laboratory of Dr. Simona Polo we identified two ubiquitin binding domains (UBDs) within the tail domain of Myosin VI (MIU and MyUb). Insertion of an alternatively spliced element between these two UBDs can lead to the formation of at least three different Myosin VI isoforms. We found that Myosin VI isoforms containing this spliced insert mainly interact with components of the endocytic machinery whereas the isoform without insert binds many nuclear interactors. Interestingly, cancer cells selectively express the short isoform, indicating that mainly nuclear functions of Myosin VI associate with oncogenesis. In the laboratory of Prof. Helle Ulrich (in collaboration with Petra Beli) I confirmed and specified the interactions of the short isoform of Myosin VI with nuclear proteins involved in fundamental genome maintenance pathways such as DNA repair, DNA replication and immune signalling. Furthermore, using cell culture experiments I was able to demonstrate functional importance for Myosin VI in the aforementioned pathways in which I show a DNA dependent Myosin VI localization on filamentous structures in the nucleus.The aim of this project is to investigate the yet poorly understood functions of Myosin VI in the nucleus but also in the cytoplasm with a focus on DNA replication and –repair as well as immune signalling. Characterization of ubiquitin-dependent processes should reveal molecular mechanisms of the Myosin VI regulated pathways. We expect novel insights on how the cytoskeleton interacts with chromatin and its influence on the dynamics of nuclear processes. Results obtained from this project should provide fundamental knowledge to understand the pathological events during oncogenesis in prostate and ovarian carcinomas and hence help to identify new molecular targets for cancer therapy.