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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SATBIOFUN (Genome editing for spatiotemporal analysis of centriolar SATellite BIOgenesis and FUNction in cellular stress responses)

Teaser

Summary

Cells contain specialized structures that perform a range of functions essential to maintaining the health of the cell. Defects in these structures or their function can manifest in a disease state. Understanding how these structures contribute to the maintenance of a healthy state allows us to learn how diseases arise and develop strategies to prevent or treat them. One such structure is the centrosome. The centrosome contributes to a number of cellular processes and serves as the base of the primary cilium, an antenna-like structure that allows cells to respond to external stimuli. Around the centrosome lie a number of protein-containing granules, termed centriolar satellites. These are involved in the movement of proteins and are crucial for normal centrosome and cilium assembly and function. Currently little is known about the full role, composition, or regulation of satellites, although mutations in genes encoding satellite proteins result in human diseases, such as ciliopathies and neurological disorders.

The central scientific goal of this project was to expand our knowledge with regard to satellite function and contribution to maintaining a disease-free state. The realization of this goal was achieved through a number of specific objectives, namely: the generation of a unique toolbox of reagents with which to study satellites; profiling of satellite protein contribution to normal satellite function; and the definition of the contribution of satellites to cell stress responses. Furthering our understanding of satellites function enhances our knowledge of how their dysregulation may lead to disease. To achieve these objectives, we used genome-editing and cutting-edge imaging techniques. This allowed us to define a novel function for satellites in the cellular response to proteostatic stress, thereby identifying a previously unexplored link between the centrosome and proteostasis fields.

In realizing our objectives, the Fellow received extensive training in a range of state-of-the-art techniques during an Outgoing Phase in Canada. The Return Phase in Ireland allowed the transfer of this knowledge to Europe, and in doing so enhanced the European research environment. The Fellow also acquired a range of complementary skills, allowing the fulfillment of the overarching objective of developing the Fellowâ€™s career to a position of professional maturity and facilitating the goal of establishing her own independent research group.

Work performed

Studying the consequences of removing a protein from a cell tells us a lot about the role that protein plays in the normal cellular function. Thus, the project initiated with the generation of a number of cell lines lacking the satellite proteins we are interested in. This was achieved by employment of the CRISPR/Cas9 genome editing system. This technique leads to the disruption of the targeted genes, meaning the protein of interested is no longer expressed. In total, the projected utilized 6 such cell lines lacking satellite proteins. Additionally, an alternative method of protein knockdown, siRNA, was also used. While these techniques were employed and optimized a number of pharmacological inhibitors, that impact a range of cellular processes, were tested for their effect on satellites. Strikingly, it was observed that inhibition of the proteasome had a profound effect on the appearance of satellites within the cell. The proteasome is a complex that the cell uses to recycle the components that make-up proteins. Therefore, the proteasome is crucial to regulating the concentration of proteins within the cell and preventing the toxic accumulation of damaged proteins. Inhibition of the proteasome leads to the collection of non-degradable proteins into a structure around the centrosome, called the aggresome. We found that satellites became entangled within the aggresome following proteasome inhibition. Further, aggresome formation was blocked in lines lacking satellite proteins. This means that satellites themselves are essential to the formation of the aggresome, which is an important observation for understanding how protein degradation is regulated. This allowed us to build a model in which satellites contribute to the movement of proteins to the aggresome. By closely examining the role of individual satellite proteins in aggresome formation, we have observed that some of the proteins are required earlier in the aggresome pathway, namely when proteins first start to aggregate within the cytoplasm. Knockdown of these proteins also prevented aggresome-like structures induced via alternative methods. This raises the tantalizing possibility that satellites play a role in protein aggregation in diseases that are characterized by the excessive accumulation of protein aggregates. We started to explore this by looking at aggregation of the protein that becomes dysregulated in Huntingtonâ€™s disease. Satellites associate with these aggregates and when disrupted reduce the size to which the aggregates can grow. These results will be exploited through collaborations we have established with research groups that are experts in neurodegenerative diseases, which are all protein aggregation disorders. Together our findings present a new model for the regulation of protein levels in cells, linking centriolar satellites to a novel activity in cellular regulation and provide insight into how satellites might contribute to disease states. These observations have been disseminated to our colleagues and collaborators through meetings and seminars, and our fields by oral presentation at international conferences. A manuscript describing this work will submitted in the coming months. The public has been made aware of our research through events both in Canada and Ireland.

Final results

Studies encompassing centriolar satellites generally reside in the centrosome field, while research focusing on aggresome formation belongs in the proteostasis field. This project has uncovered a novel link unifying these fields, placing satellites in the regulation of protein degradation. The efficiency of the proteasome to recycle proteins is known to decline with age and aggregates of misfolded proteins also compromise its function. Both these factors are important in neurodegenerative diseases, such as dementia, Parkinsonâ€™s, Huntingtonâ€™s and Alzheimerâ€™s, which are characterized by the accumulation of toxic protein aggregates. As aggresome formation represents a specialized cellular response to the failure of the proteasome machinery, our work furthers our understanding of the pathogenicity of these conditions. Indeed, many of the defective aggregation-prone disease-causing proteins identified to date have specific motifs that target them to aggresomes. Through this study we have identified a novel role for satellites in the processing of protein aggregates, thereby providing insight into how cells prevent disease states from occurring. This knowledge allows us to test the specific role of satellites in neurodegenerative disease model systems with the potential to prevent those diseases from evolving. Increasing age is the biggest risk factor for the development of these disorders, hence with an aging population they become more prevalent. Understanding how cells process protein aggregates, how this changes with age, and how aggregates reach toxic levels, is fundamental to developing new therapies for the prevention and treatment and of neurodegenerative diseases.