Multiscale nuclear mechanobiology within the skin: from biophysical cues to epigenetic effects
Lead Research Organisation:
Queen Mary University of London
Department Name: Blizard Institute of Cell and Molecular
Abstract
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.
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.
Technical Summary
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 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.
Planned Impact
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.
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.
People |
ORCID iD |
John Connelly (Principal Investigator) | |
Nuria Gavara (Co-Investigator) |
Publications
Keeling MC
(2017)
Actomyosin and vimentin cytoskeletal networks regulate nuclear shape, mechanics and chromatin organization.
in Scientific reports
Pundel OJ
(2022)
Extracellular Adhesive Cues Physically Define Nucleolar Structure and Function.
in Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Gonzalez-Molina J
(2018)
Extracellular fluid viscosity enhances liver cancer cell mechanosensing and migration.
in Biomaterials
Zhang X
(2020)
Ezrin Phosphorylation at T567 Modulates Cell Migration, Mechanical Properties, and Cytoskeletal Organization.
in International journal of molecular sciences
Almeida FV
(2019)
High-Content Analysis of Cell Migration Dynamics within a Micropatterned Screening Platform.
in Advanced biosystems
Flores LR
(2019)
Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour.
in Scientific reports
Connelly JT
(2021)
Research Techniques Made Simple: Analysis of Skin Cell and Tissue Mechanics Using Atomic Force Microscopy.
in The Journal of investigative dermatology
Laly AC
(2021)
The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction.
in Science advances
Sliogeryte K
(2019)
Vimentin Plays a Crucial Role in Fibroblast Ageing by Regulating Biophysical Properties and Cell Migration.
in Cells
Description | This research project employed micro-patterned substrates to understand how biophysical forces transmitted through cell-matrix and cell-cell adhesions impact nuclear mechanics and chromatin remodelling. Our findings reveal that cell shape is a potent regulator of global changes in nuclear architecture and that cell-matrix adhesions actively deform the nucleus via the F-actin cytoskeleton, while cell-cell adhesions and the keratin network of intermediate filaments protect the nucleus from deformation through cytoskeletal remodelling and stress shielding. Forces transmitted to the nucleus influence remodelling of the nuclear lamina, chromatin condensation, and nucleoli size and number. Profiling of downstream changes in chromatin remodelling and gene expression using next-generation sequencing methods have identified key signalling pathway and networks regulated by these biophysical cues. Through these studies we have also developed new imaging and biophysical methods (atomic force microscopy, AFM) to directly assess the mechanical properties of the keratin cytoskeleton. Finally, we have discovered that these normal mechano-sensing mechanisms are disrupted in cancer cells and blistering skin diseases. |
Exploitation Route | The genes and signalling pathways identified here could represent important therapeutic targets for skin diseases affecting cell mechanics and adhesion, such as blistering diseases, kerataderma, and even squamous cell carcinoma. Also, the new AFM methods developed here could be shared with other researchers interested in the mechanics of cells and the cytoskeleton. |
Sectors | Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | The methods and procedures for generating micro-patterned surfaces are used as laboratory practical for the MSc Regenerative Medicine programme at Queen Mary, and the PDRA working on this project has carried laboratory demonstrations for these practicals. Thus, this project has so far had an educational impact. |
First Year Of Impact | 2018 |
Sector | Education,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal |
Description | Biophysical regulation of nucleolar structure and function in cellular senescence and ageing |
Amount | £554,318 (GBP) |
Funding ID | BB/X006972/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2023 |
End | 03/2026 |
Description | Investigating the impact of EBS-causing keratin mutations on epigenetic gene regulation |
Amount | £33,589 (GBP) |
Funding ID | Connelly 1 |
Organisation | DEBRA International |
Sector | Charity/Non Profit |
Country | Austria |
Start | 05/2023 |
End | 01/2024 |
Description | Understanding the mechanobiology of senescence and ageing |
Amount | £126,098 (GBP) |
Funding ID | RPGF1810\82 |
Organisation | The Dunhill Medical Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2019 |
End | 06/2021 |
Description | Invited talk at Brunel University meeting 'Cells Under Pressure' |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | I presented our most recent research on the role of mechanical and biophysical cues in nuclear organisation and gene regulation. The talk stimulated questions from the audience and discussion afterwards. This engagement led to new understanding of nuclear mechanotransduction by audience members and potential collaborative links. |
Year(s) Of Engagement Activity | 2020 |
Description | Invited talk at Epigenetics symposium at the annual ESDR meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Dr. John Connelly gave an invited talk at the annual European Society for Dermatological Research meeting. He spoke about the role of biophysical cues in the epigenetic regulation of keratinocyte behaviour. |
Year(s) Of Engagement Activity | 2017 |
Description | Invited talk at the Annual ESDR Meeting - Epigenetics Symposium |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I presented recent findings on the role of mechanical and biophysical cues in the regulation of nuclear architecture and chromatin remodelling. The talk stimulated discussion and potential collaborations with audience members. |
Year(s) Of Engagement Activity | 2019 |
Description | Presentation at the European Intermediate Filaments meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | John Connelly gave an invited talk describing the role of the keratin cytoskeleton in nuclear mechanotransduction. The audience was approximately 150 scientists with expertise in the field of intermediate filaments. The purpose was to disseminate the findings of our research to other working in this area. |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.moca.rwth-aachen.de/euroif2021/index.html |
Description | Research Seminar at University College London |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Professional Practitioners |
Results and Impact | I delivered a research seminar to the Physics of Living Systems group at UCL and presented work from the BBSRC funded project. |
Year(s) Of Engagement Activity | 2019 |