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

非常规肌球蛋白是肌动蛋白为基础的分子马达蛋白，负责细胞内囊泡运输、结构维持和力产生过程。肌球蛋白VI代表肌球蛋白超家族的独特成员，因为它的运动方向与迄今为止分析的所有其他肌球蛋白相反。它参与各种信号传导途径（例如内吞作用、分泌、自噬等）。而其失调通常与人类疾病有关。在前列腺癌和卵巢癌中，肌球蛋白VI属于最严重的上调基因。除了细胞质定位，肌球蛋白VI也被发现存在于细胞核中，尽管其核功能的特点不佳。在我的第一个博士后在西蒙娜波罗博士的实验室，我们确定了两个泛素结合结构域（UBD）内的尾部结构域的肌球蛋白VI（MIU和MyUb）。在这两个UBD之间插入选择性剪接元件可导致至少三种不同肌球蛋白VI同种型的形成。我们发现，肌球蛋白VI亚型含有这种剪接插入主要相互作用的内吞机制的组件，而没有插入的亚型结合许多核相互作用。有趣的是，癌细胞选择性表达短亚型，表明肌球蛋白VI的主要核功能与肿瘤发生有关。在海勒乌尔里希教授的实验室（与佩特拉·贝利合作），我证实并详细说明了肌球蛋白VI的短亚型与参与基本基因组维持途径（如DNA修复、DNA复制和免疫信号传导）的核蛋白的相互作用。此外，使用细胞培养实验，我能够证明功能的重要性，肌球蛋白VI在上述途径中，我显示了一个DNA依赖肌球蛋白VI的定位在丝状结构在nuclear.The项目的目的是调查尚未了解的功能肌球蛋白VI在细胞核中，但也在细胞质中的DNA复制和修复以及免疫信号。泛素依赖性过程的表征应揭示肌球蛋白VI调节途径的分子机制。我们期待新的见解，细胞骨架如何与染色质相互作用，其对核过程的动态影响。从这个项目中获得的结果应该提供基本的知识，以了解在前列腺癌和卵巢癌的肿瘤发生过程中的病理事件，因此有助于确定新的分子靶点的癌症治疗。

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.