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Regulation of midbody formation and function by phosphorylation

Reference	BB/W01372X/1
Principal Investigator / Supervisor	Dr Pier Paolo D'Avino
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Pathology
Funding type	Research
Value (£)	652,985
Status	Current
Type	Research Grant
Start date	01/09/2022
End date	31/08/2025
Duration	36 months

Abstract

The mechanics and directionality of cell division is regulated by reversible post-translational modifications, like phosphorylation. Although significant advances have been made in understanding how phosphorylation controls chromosome alignment and segregation, much less attention has been devoted to the regulation of cytokinesis. Here we propose to study how phosphorylation regulates the formation and functions of the midbody, an organelle that forms between the two dividing cells during cytokinesis. The midbody is crucial for the abscission of the daughter cells and has been implicated in various post-mitotic processes, including cell fate, proliferation, apical-basal polarity, cilium and lumen formation, cancer and microcephaly. Although initial studies have revealed that phosphorylation plays a key role in regulating the activity, function, and associations of midbody proteins in time and space, our understanding of the mechanisms that regulate its formation and functions are still very limited. We propose to employ a multi-system approach, involving a combination of affinity purification, quantitative proteomics, bioinformatics, and high resolution multi-dimensional imaging, to investigate how phosphorylation controls the dynamics, stability and interactions of 6 key midbody components. We will use cell lines harbouring genes tagged with a fluorescent protein by gene editing to identify the interactomes and analyse the dynamics of these 6 proteins in normal conditions and after perturbing cytokinesis with inhibitors of mitotic kinases and phosphatases. Computational analysis of these results will lead us to design specific mechanistic hypotheses that we can then test using a combination of cell biology and biochemical experiments. Our findings will unravel the molecular mechanisms by which phosphorylation regulates midbody formation and functions, thus helping us understand how this organelle mediates so many important cellular and developmental functions.

Summary

All biological processes are controlled and executed by a class of molecules, the proteins, whose functions are regulated in both space and time by post-translational modification (PTMs). Reversible PTMs, such as phosphorylation, are essential for the proper activity of many signalling pathways and networks that control almost every cellular and developmental process. Phosphorylation and de-phosphorylation are mediated by opposing enzymes: kinases, which add phosphate groups to their target substrates, and phosphatases that instead remove them. The addition or removal of these phosphate groups regulate the activity, localisation and association of proteins. Recent advances in the field of proteomics have led to the identification of many proteins that are regulated by phosphorylation in different cellular contexts. However, in vivo functional studies have revealed the existence of complex interplays between kinases and counteracting phosphatases that are largely unknown. Therefore, understanding how protein regulation by kinases and phosphatases regulates the function of proteins and protein complexes in specific biological events represents one of the main future challenges in bioscience research. In addition, as phosphorylation is often altered in many human diseases, detailed knowledge of kinases and phosphatases cross-regulation and functions in normal conditions could provide a framework for revealing how phosphorylation mechanisms and pathways are altered in pathological conditions and aid the design of future clinical therapies. The goal of our research project is to understand how phosphorylation controls the formation and functions of an organelle, the midbody, that forms between the two daughter cells at the end of cell division. The midbody is essential for completion of cell division and has been implicated in various post-division processes, including cell fate, pluripotency, apical-basal polarity, tissue organisation, cell proliferation, brain development, and cilium and lumen formation. In addition, midbody proteins have been linked to human diseases, including cancer and impaired brain development (microcephaly). We plan to employ an interdisciplinary approach involving a combination of innovative and advanced methodologies, including gene editing, quantitative proteomics, bioinformatics, and high resolution multi-dimensional imaging, to unravel how phosphorylation regulates the function, dynamics and association of midbody proteins. Our findings will provide key insights into the regulation of midbody proteins and help understand how this organelle mediate so many important functions in cells. Furthermore, our results will pave the way for the development of targeted therapies for human diseases like cancer and some neurological conditions. Our study will have a far-reaching impact in many research areas beyond the study of cell division and including medical disciplines.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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