As a non-membrane bound organelle, the assembly of centrosomes must be driven by PPIs. These interactions are likely modified by highly regulated changes in protein binding affinity in a cell-type and cell-cycle dependent manner, which can in turn modulate centrosome behavior and function. We have focused on identifying PPIs among a core set of conserved centrosome proteins, classified as proteins of the centriole, the PCM and regulatory kinases. Previous high-throughput and small-scale interactions studies have suggested that there might be a limited number of PPIs among centrosome proteins and have raised the possibility that few interactions are used to construct a simple, ultimately static structure. Our data does not support this simplistic view of the centrosome. We have uncovered a large number of direct PPIs, dramatically expanding our understanding of the centrosome interaction landscape. This highly interconnected landscape suggests a much more complex centrosome assembly and regulatory process, which we suggest can be leveraged to perform a variety of specialized tasks dictated by a broad spectrum of cellular requirements. In previous years we have focused our attention on Pericentrin-Like-Protein (PLP), Asterless (Asl), Cep135, and the critical centriole duplication kinase Plk4. In the past year, we have added the protein Ana2 and Cnn in collaboration with the labs of David Agard and Gregory Rogers. These studies have uncovered important regulatory mechanisms related to centriole duplication and PCM assembly. However, our main focus for the past year has been to determine how a proximal end of the centriole is established, and for what purpose. Interestingly, the pericentriolar material (PCM), which organizes microtubules is frequently positioned at the proximal end of the centriole. How PCM is specifically localized to the proximal end, and if this localization has functional significance, remains largely unexplored. We have used the centrioles of spermatocytes in the Drosophila male germline, which grow to almost 2 microns, to interrogate how the Drosophila Pericentrin ortholog (PLP) is localized to the proximal end of the centriole, and test if this localization is critical for function. We find that the localization of PLP to the proximal end is achieved by limiting the availability of PLP protein to the earliest phases of centriole growth, before centriole elongation. Furthermore, plp transcript is also rapidly lost prior to centriole elongation. Exogenous expression of PLP under the control of promoters that specifically express during the elongation period, result in the deposition of PLP along the entire length of the centriole; promoters that express prior to elongation behave as wild type. Interestingly, during meiosis, centrioles with PLP inappropriately along their entire length also have PCM recruited along entire length, indicating that PLP is sufficient to instruct PCM localization. To determine if the positioning of PLP and PCM at the proximal end has a functional consequence, we followed centrioles as spermatocytes developed into spermatids. Through live cell imaging in developing testes, and careful staging of fixed tissue, we find that centrioles with PLP and PCM along the entire length are positioned such that the centriole is laterally associated with the nucleus as opposed to the normal proximal-end docking to the nucleus. This, to our knowledge, is the first demonstration that the proximal localizing of PCM serves a purpose to ensure proper centriole-nucleus attachment in spermatids. Furthermore, our preliminary studies indicate that this improper centriole docking results in a major reduction in properly formed sperm. We are now testing the impact of mispositioning PCM on overall animal fertility. By combining our interactome with in vivo experimental evidence in Drosophila, we demonstrate how large interaction information can lead to in depth mechanistic insight into macromolecular assemblies. Integrating protein localization, dynamics and functional data with direct PPI information and mutant analysis, we show that interactions can predict inter- and intra-molecular architecture, identify kinase substrates and uncover regulated interactions within the centrosome. Understanding the interaction landscape of the centrosome is a critical foundation needed to gain an understanding of the molecular basis for human diseases caused by dysfunction of centriole, centrosome and cilia proteins. The diversity of centrosome-related diseases, such as microcephally, dwarfism, polycystic kidney disease and many others, can be best explained by loss of specific PPIs, rather than a simple complete loss of protein function stemming from a null mutation. We believe that our study will also guide new avenues of centrosome research that focus on the in-depth understanding of the diverse functions of these proteins, and could serve as a framework to explore other complex cellular processes.
Showing the most recent 10 out of 13 publications
