Multiscale nuclear mechanobiology within the skin: from biophysical cues to epigenetic effects

The epidermis of the skin forms an essential physical barrier to the external environment, and it is continuously exposed to complex biomechanical forces. While it is well known that mechanical and biophysical cues regulate diverse cellular functions, such as growth, migration, and survival, the molecular level mechanisms by which cells within the skin sense these forces remains poorly understood. As the nucleus is the central organelle within which DNA is packaged and this internal structure defines specific patterns of gene expression, we hypothesise that the nucleus is a major mechano-sensing element within the cell: Mechanically induced changes in nuclear shape influence the internal structure and transcription of genes. Indeed, our preliminary data indicate that keratinocytes exposed to defined physical cues display changes in nuclear size and shape, and these changes in nuclear morphology correlate with altered DNA structure.

The overall objective of the proposed project is to understand how forces are transmitted from the external environment to the nucleus and to determine the subsequent effects on nuclear structure, gene expression, and cell function within the epidermis of the skin. We will use advanced biophysical and imaging techniques to apply forces to single cells, and systems biology methodologies to analyse the changes in DNA structure and gene expression. In addition, we will test the role of internal cellular structures, such as the cytoskeleton, to gain mechanistic insight into these processes. Finally, we will investigate the influence of nuclear mechano-sensing in more complex 3D models of human skin.

The successful completion of this project will provide fundamental and significant insights into how cells sense mechanical forces, in particular the role of the nucleus. This knowledge will advance our understanding of normal skin physiology and mechanical function. Additional impacts of the proposed project include the development of new biophysical tools and technologies, building new collaborations with academics and industry, and multi-disciplinary training for the scientists involved. Future studies following on from this work could involve investigation of additional aspects of nuclear structure and examining the role of these mechanotransduction pathways in skin diseases such as blistering, scarring, or cancer.

Mechanical and biophysical forces are major regulators of fundamental cellular processes, such as growth migration, differentiation, and survival. While the nucleus is believed to be a central mechano-sensing element within the cell, the mechanisms by which forces are transmitted to the nucleus and converted into biochemical and genetic signals remain poorly understood. Our preliminary data indicate that biophysical cues affect nuclear size and shape in human keratinocytes, and these changes in morphology correlate with altered chromatin structure and condensation. We therefore hypothesise that extrinsic mechanical forces regulate gene expression and cell function within the epidermis of the skin through direct biomechanical effects on nuclear structure and chromatin remodelling.

The aims of the proposed research project are to determine the mechanisms of force transmission to the nucleus and to investigate the downstream changes in chromatin remodelling, gene expression, and keratinocyte function. Atomic force microscopy will be used to apply controlled forces to single cells, while live fluorescence imaging will be used to map the pattern of force transmission and nuclear deformation. In addition, genomic methods including ChIP-seq and RNA-seq, will be used to characterise specific changes in chromatin remodelling and gene expression induced by biophysical cues. The roles of cytoskeletal structures involved will be examined using chemical inhibitors and genetic knock downs, and the physiologic impact of mechanically-induced changes in chromatin remodelling will be explored using 3D models of human skin exposed to stretch. Together, these studies will provide fundamental insights into the mechanisms of nuclear mechano-sensing and the impact on cell and tissue function. These findings will have important implications for our understanding of normal skin physiology, epigenetic mechanisms of gene regulation, and cellular biomechanics.

The proposed research project has the potential to deliver economic and social impacts through a variety of different mechanisms. These include technology and tool development for the imaging and biotechnology sectors, the advancement of training and public engagement, and over the long term, improved human health and well-being.

1. Industrial impact: This proposal will modify AFM-based instrumentation and will also use image processing algorithms for morphological characterisation of cells and their nuclei. In both activities, we will use commercial systems and combine them with our own analysis algorithms. Commercial AFM systems designed for cell biology work (Catalyst by Bruker, MFP3D by Oxford Instruments, Nanowizard3 by JPK Instruments) are equipped with pre-programmed measurement routines, so that inexperienced users can easily perform measurements without the need for troubleshooting or optimisation. We believe equipping an AFM with an easy-to-use pre-programmed measurement routine for mechanical stimulation of cells and the accompanying data processing algorithms could be equally included in existing data analysis toolboxes. Dr Gavara is already in conversations with two AFM companies (Bruker and JPK) and we are on track towards formalising collaboration agreements during the current year. Image processing algorithms developed for cell and nuclei morphological characterisation could also be included in high-throughput commercial imaging systems. New applications for the epigenetic methods used here could be taken up and marketed by biotech companies, such as Active Motif and Diagenode. Over the long-term, insights into the downstream genes and cellular functions regulated by mechanical forces could lead to the development of new therapies and treatments by pharmaceutical, personal care, or biotech companies.

2. Education and training: This interdisciplinary project will be an excellent training opportunity for the PDRAs involved. As each will have a distinct set of skills and expertise (e.g. bioscience vs physics), they will learn from each other and gain new knowledge and experience. This training will lead to the development of multi-disciplinary researchers and help advance the careers of these scientists. In addition, Biomedical Engineering MEng students will participate in small aspects of this research programme, as part of their design and development group project. The students will acquire a variety of skills, including algorithm design, code writing, and instrument control, as well as exposure to molecular and cell biology. This skillset will likely help them make a contribution to the competitiveness of the UK once they incorporate into the graduate job market. Similar projects will be available to the 3rd year Biomedical Science and Regenerative Medicine MSc students.

3. Public engagement: Through outreach and public engagement activities, we plan to raise awareness about the role of mechano-biology in normal cell and tissue functions, as well as how biomechanics can influence disease processes. Working with the Centre of the Cell at Queen Mary, these activities will help inform the general public on current areas of biomedical research, potential benefits, risks, and limitations of this work.

4. Health and well-being: A long-term goal of this research project will be to understand the biological function of mechanically-driven changes in chromatin remodelling and gene expression. These regulatory mechanisms may have important implications for normal skin function and influence the pathogenesis of conditions with altered biomechanics, such as genetic blistering diseases, wound healing, and cancer. Thus, our research may lead to the identification of new therapeutic targets or prognostic markers, which would ultimately improve human health and well-being. To deliver this impact we will engage both with industry and clinicians to identify new areas for future research projects.