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How does the desmosome-actin crosstalk regulate desmosome function?

Reference	BB/X008827/1
Principal Investigator / Supervisor	Professor Christoph Ballestrem
Co-Investigators / Co-Supervisors	Professor David Garrod
Institution	The University of Manchester
Department	School of Biological Sciences
Funding type	Research
Value (£)	554,388
Status	Current
Type	Research Grant
Start date	01/04/2023
End date	31/03/2026
Duration	36 months

Abstract

Desmosomes are cell-cell junctions linked to intracellular intermediate filaments (IF). They adopt hyper-adhesion that assists tissues, e.g. epidermis and heart muscle, to resist shear. In development and wound repair, tissue reorganisation involves both formation and downregulation of desmosomes. Our pilot Bio-ID data suggest the involvement of actin-binding proteins in these processes. Additional new Bio-ID data show a substantial change in the interactome of the desmosomal protein plakophilin 2 (Pkp2) upon inhibition of actomyosin function. On this basis we hypothesise that the regulation of desmosomal adhesion and the signalling involves crosstalk between actin and desmosomes. Our major question is: How does the actin-desmosome crosstalk regulate desmosome adhesion? This question divides as follows: 1) What are the mechanisms of the actin-desmosome crosstalk and how do they regulate desmosomes? We will use knockdown/out technology together with advanced fluorescence microscopy (FRAP and photoactivation) to study how actin-associated proteins are involved in the regulation of desmosomes. To identify how proteins interact, we will use a mitochondrial targeting system that allows the screening of protein interactions. The use of mutants in cells will reveal how these interactions contribute to the life cycle of desmosomes. 2) How does Pkp2 contribute to desmosome regulation and signalling? Using molecular biology techniques combined with imaging we will examine how Pkp2 is recruited to desmosomes, how it interacts with binding partners, and how these interactions and the actin-cytoskeleton contribute to desmosome stability. BioID and imaging of Pkp2 in different cellular compartments under varying conditions will provide detailed understanding where interactions Pkp2 take place and how they contribute to desmosome signalling. Insights from these studies will further our understanding of development may lead to novel disease therapies.

Summary

Cells in stress-exposed tissues, e.g. heart muscle and coverings of body surfaces, are bound together by tiny structures called desmosomes, aberrant function of which causes diseases such as heart failure, defective wound healing, cancer spread and blistering diseases of the skin and oral cavity. Desmosomes are also important for normal development, where they stabilise developing tissues. It is therefore essential to understand how desmosome function is regulated. We have shown that an important factor contributing to tissue toughness is that the ability of desmosomes to adopt a highly adhesive state known as hyper-adhesion. Hyper-adhesion is important for tissue strength, but also locks cells together restricting their movement. During wound healing, epidermal cells migrate to close the wound. The invasive spread of cancer cells also requires cell migration and in development cell movement generates the correct architecture of tissues. When cells move and grow to establish cell sheets, they form new desmosomes that mature to become highly adhesive. When cell sheets are wounded they rapidly lose hyper-adhesion and downregulate desmosomes by internalising them. Little is known about how desmosomes assemble, change their adhesive state and how they are downregulated when this is needed. Desmosomes have a characteristic structure made up of a few components and we have found that most of these components are stably integrated into the structure. However, one of them, called plakophilin (Pkp), moves rapidly from the periphery to central parts of the cell and vice versa. We think that this dynamic behaviour serves to transmit information (signals) within the cell and leads to changes in cell behaviour. Normally desmosomes appear at the junctions between cells but studies have shown whole desmosome inside cells, as though one cell has "eaten" the desmosome! We have now induced cell separation in culture and shown that they do indeed engulf whole desmosomes. This isexciting because it enables us to investigate the mechanism behind a process that occurs in normal and diseased tissues. Desmosome engulfment resembles a process called phagocytosis whereby cells of the immune system engulf extracellular particles, e.g. bacteria. Phagocytosis requires active contractile activity by the engulfing cell so as to surround the particle and draw it inside. Such contractility requires the action depends upon filamentous proteins called actin and myosin, similar to those involved in muscle contraction. Functional actin and associated proteins are also important for desmosome regulation. Desmosomes are normally internally linked to other filaments called intermediate filaments (IF), rather than actin. Desmosomes link IF from cell-to-cell by, forming a scaffolding that gives strength to tissues. However, IF possess nor contractile activity Hence the question that arises of how actins link up to desmosome to regulate their function when contractility is required? We have pilot data showing that a number of proteins known to interact with actin are very close to desmosomes and we think that these are involved in regulation of desmosome adhesion and engulfment. Our data also suggest that the actin cytoskeleton influences signalling by Pkp. We will use state-of the-art microscopy to study how desmosomes become associated with the contractile machinery as they assemble and switch from hyper-adhesion to engulfment, and mass spectrometry to identify how actin binding proteins are involved in regulating desmosome function. Finally, we will use molecular cell biology to determine the role Pkp in this process and in signalling cell behaviour. Our results will both further our understanding of normal development and provide a basis for new therapies for major health problems such as chronic wounds, some types of heart failure, skin blistering diseases and, potentially, for limiting the spread of cancer.

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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