Deciphering the cellular roles of pericentriolar satellites using an inducible degron system.

中心体是细胞中富含蛋白质的细胞器，参与细胞分裂、信号传导和纤毛的形成。在增殖细胞中，中心体在细胞分裂过程中促进有丝分裂纺锤体的组装和染色体正确地分裂成子细胞。在静止（非增殖）细胞中，中心体为原纤毛的组装提供模板，原纤毛是存在于质膜中的一种感觉和信号细胞器。因此，中心体的数目、组成、位置和功能必须受到严格的调控，不仅在增殖细胞中如此，在静止细胞中也是如此。中心周围卫星是聚集在中心体周围的富含蛋白质的小颗粒。它们与中心体和纤毛的调节有关，但它们的确切作用尚不清楚。一个特别有趣的可能性是，心泡周围卫星整合细胞信号来决定是否以及何时将中心体转化为初级纤毛。到目前为止，卫星功能的研究是通过耗尽必要的卫星支架PCM1来进行的。尽管这种方法提供了信息，但它在几天后实现了PCM1损耗，因此不适合研究卫星损耗的直接影响。本提案的主要目标是建立一种新的实验工具，可以快速和可逆地去除PCM1蛋白及其相关卫星。该方法将包括在PCM1蛋白上插入一个小标签。在添加特定化合物后，该标签将被细胞自身的降解机制识别。利用这项技术，我将能够解决卫星如何调节中心体和纤毛的组装，以及它们在增殖和静止细胞中的维持。此外，我将研究这些功能如何在纤毛和非纤毛细胞类型之间有所不同。超分辨率和活细胞显微镜以及生化方法将在这些研究中使用。研究中心周围卫星对中心体和纤毛功能的调节作用将有助于我们更好地了解它们在神经发育障碍和癌症中的作用。

Centrosomes are protein-rich organelles in the cell that are implicated in cell division, signalling and cilia formation. In proliferating cells the centrosome promotes mitotic spindle assembly and the correct partitioning of the chromosomes into daughter cells during cell division. In quiescent (non-proliferating) cells the centrosome templates the assembly of the primary cilium, a sensory and signalling organelle present in the plasma membrane. Centrosome number, composition, location and function must therefore be tightly regulated not only in proliferating but also in quiescent cells. Pericentriolar satellites are small, protein-rich granules that cluster around the centrosome. They have been implicated in the regulation of both centrosomes and cilia, but their precise contribution remains unclear. A particularly interesting possibility is that pericentriolar satellites integrate cellular signals to decide whether and when to convert centrosomes into primary cilia. So far, satellite function has been studied through depletion of the essential satellite scaffold, PCM1. Although informative, this method achieves PCM1 depletion after several days and is thus ill-suited to study the immediate effects of satellite loss. The key objective of this proposal is to establish a new experimental tool that allows rapid and reversible removal of the PCM1 protein and its associated satellites. The method will involve the insertion of a small tag onto the PCM1 protein. This tag will be recognised by the cells own degradation machinery after addition of a particular compound. Using this technology, I will be able to address how satellites regulate centrosome and cilia assembly as well as their maintenance in proliferating and quiescent cells. Additionally, I will investigate how these functions may differ between ciliated and non-ciliated cell types. Super-resolution and live-cell microscopy as well as biochemical approaches will be used in these studies. Investigating the regulatory role of pericentriolar satellites to centrosome and cilia function will help us to better understand their contribution to neurodevelopmental disorders and cancer.