Bridging the gap between the mechanical and metabolic activity in cell-ECM interactions

San Felix Garcia-Obregon, Ana (2023) Bridging the gap between the mechanical and metabolic activity in cell-ECM interactions. PhD thesis, University of Glasgow.

Abstract

Cells are in constant communication with their surroundings. This is possible by establishing focal adhesions (FAs) formed by the attachment of transmembrane proteins known as integrins, with proteins from the extracellular matrix (ECM) (e.g. fibronectin (FN) or collagen). The ECM is the natural scaffold of the cells within the tissues. It is a three-dimensional scaffold generated by molecules secreted by the cells themselves. The properties of the ECM vary among the different tissues, which influences different cellular behaviour and differentiation (1,2). For instance, bone presents a stiffer ECM than the brain, which has one of the softest matrices within the body (1). The ECM is the direct link of the cell with the environment and every piece of information gathered from the FAs plays an important role in cellular fate (1).

Mesenchymal stem cells (MSCs) are multipotent cells that have self-renewal capacity and can differentiate into different tissues such as bone, adipose tissue or cartilage (3). In the last decade, those cells have gained high importance in research, especially in regenerative medicine since they can be used in vivo and in vitro (4). The type of tissue they differentiate into depends on how they sense the matrix, among other things. It has been shown that on stiff surfaces they differentiate into harder tissues, such as bone, while on softer matrices they differentiate into a less rigid tissue as is the case of adipose tissue (1,5). Therefore, it is important to understand cell-ECM interactions and what intracellular changes occur upon these interactions.

Multiple studies have demonstrated the implications of matrix mechanical properties in cellular mechanical and metabolic activity (6,7). In general, cells on stiff matrices can form mature FAs where all the proteins involved are gathered to resist the ECM tension and attach (8). Cells form their actin-cytoskeleton and contract to generate high intracellular tension. This is necessary to exert forces through the FAs and compensate for that extrinsic tension (9). This increase in cellular contractility and tension triggers different pathways (e.g. YAP/TAZ) and leads to nuclear flattening, which favours the translocation of transcriptional factors into it such as YAP to the nucleus (10–12). Once in the nucleus YAP binds to the DNA and starts the expression of different proteins involved in cellular mechanics and metabolism (13). All this implies high energy investment from the cell. The main source of energy is ATP, which is generated during cell respiration. Thereby, on stiff surfaces cellular respiration increases to supply that energy demand (13). On the contrary, on soft surfaces FAs maturation, cell contractility, nuclear flattering and YAP nuclear translocation decreases (11,12). Under these circumstances, cells do not consume as much energy as on stiff surfaces, hence cellular respiration decreases as it does ATP production and consumption.

We hypothesise that cells require mechanical and metabolic energy during ECMcell interactions to generate forces and create an adaptive response. This work aims to bring together how cells behave from mechanical and metabolic perspectives during their attachment, proliferation, migration and differentiation, in relation to the matrix properties, in particular depending on matrix stiffness and degradability. This will provide a better understanding of how microenvironmental cues can be used to control cell fate, enhancing studies in multiple fields e.g. tissue regeneration or drug testing. In order to do this, cells were seeded on full-length FN-PEG and polyacrylamide (PAA) hydrogels of different stiffnesses. Traction Force Microscopy (TFM) was used to study cellular force generation on different surfaces and YAP nuclear translocation was followed by immunostaining. Cellular respiration rate, metabolites, and ATP generation were analysed to complete the cellular mechanobiological activity studied with TFM. Also, different metabolic pathways such as AMPK, NAD+/NADH or ATP/ADP were studied using ratiometric sensors. Blebbistatin was incorporated to demonstrate the importance of cell contractility in cellular response. This study concludes that cellular mechanical and metabolic activity are not independent of one to another and they are influenced by the properties of the matrix. These finding contribute to get a better understanding of cellular behaviour and, hence, develop optimal scaffolds that can be used in tissue regeneration.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology; T Technology > T Technology (General)
• Colleges/Schools: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Supervisor's Name: Salmeron-Sanchez, Professor Manuel and Dalby, Professor Matthew
• Date of Award: 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2023-83730
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 21 Jul 2023 14:12
• Last Modified: 21 Jul 2023 14:16
• Thesis DOI: 10.5525/gla.thesis.83730
• URI: https://theses.gla.ac.uk/id/eprint/83730