Pericentriolar satellites (PS) are electron dense granules surrounding the centrosome, the major microtubule-organizing centre in eukaryotic cells. In cycling cells the centrosome promotes spindle assembly and the faithful execution of mitosis. In non-cycling cells it is involved in forming the cilium, a plasma membrane-resident organelle, which mediates crucial signalling pathways in development and tissue homeostasis. PS are thought to contribute to centrosome formation, through the microtubule-dependent transport of centrosome components, and they are involved in ciliogenesis and stress response. Moreover, several proteins that localize to PS are mutated in human ciliopathies and neurodevelopmental disorders. The precise roles of PS in the various molecular pathways and diseases are however poorly understood, in part due to the limited knowledge of their composition.

In the first part of my study I performed a comprehensive analysis of the pericentriolar satellite proteome. This was achieved by sucrose sedimentation of PS, combined with affinity purification of a key PS component, PCM1. To eliminate contamination by centrosomes, the PS proteome was determined from wild-type cells as well as from two cell lines genetically engineered to lack centrosomes. Mass spectrometry identified 170 PS components including most of the previously described PS proteins, confirming the validity of the approach. Having determined the proteomic composition of PS from DT40 cells, I then performed validation studies both in chicken and human cell lines.

In the second part of my study, I aimed to use the list of PS proteins to uncover new biological roles for pericentriolar satellites. I devised two distinct approaches to gain functional insights. First, I generated a cell line lacking PCM1 as a tool to study the role(s) of PS and PS components. Second, I performed loss-of-function studies on a set of new PS proteins to determine their function(s) in maintaining the canonical PS distribution and in forming primary cilia.