Mechanisms of mechanical regulation of stem cell fate

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The emergent field of mechanobiology has established that cell behaviours are influenced by the mechanical properties of the surrounding environment. A key consequence of this influence is that cell fate can be directed by the microenvironment: human mesenchymal stem cells (huMSCs), for example, adopt an adipogenic lineage (i.e. form fat tissue) when cultured on soft materials, but are osteogenic (i.e. form bone tissue) on stiff materials. The well-characterised stiffness sensitivity of huMSCs has advantages for regenerative medicine, where the ability to direct lineage by mechanical stimulation has sparked investigations into potential applications for tissue engineering. The principles of cellular mechanosensing, though, have more general and fundamental biological importance: in embryogenesis and development, tissues must stiffen to enable them to be robust to their functions; in healthy tissues, mechanical homeostasis must be maintained; and in ageing and diseases such as fibrosis, a break-down in mechanical homeostasis can contribute to pathology.Forces are transmitted from the microenvironment into cells (and vice versa) through integrin adhesion complexes (IACs) embedded in the cell membrane. These complexes bind to proteins in the extracellular matrix (ECM) and interface with the structural proteins of the cytoskeleton. Within the cell, the cytoskeleton is in turn tethered to the nucleus by the LINC (linker of nucleo- and cytoskeleton) complex, which spans the nuclear membrane and is attached to chromatin via the nuclear lamina. There is therefore considered to be a continuous conduit of mechanical linkage between the microenvironment and the cellular centre of transcriptional regulation. The process of converting mechanical stimulation into biochemical signalling is referred to as mechanotransduction. As the field of mechanobiology has become established, a number of key mechanotransduction pathways have been identified; however, these have typically been identified through the activities of single protein entities. Here, we propose a systematic and integrated investigation into protein interactions across the entire mechano-transmission pathway, applied to examine huMSCs as they respond to changes in environmental stiffness. A combination of hypothesis-led and synthetic, global approaches will enable us to address the following objectives:(a) Using a proximity labelling approach, coupled to mass spectrometry proteomics, we will produce a catalogue of the proteins that interact with each component part of the cellular mechano-transmission pathway (including IACs and the LINC complex).(b) We will then quantify how protein interactions with component parts of the cellular mechano-transmission pathway are altered in huMSCs cultured on soft, intermediate and stiff materials. This will enable us to build a candidate list of protein interactions involved in mechanosensing.(c) We will test the importance of the identified protein interactions by introducing mutations to remove them or inhibit their activity. Consequences of impaired mechano-sensitivity will be assessed by determining whether huMSCs are still able to undergo stiffness-directed commitment to adipo- and osteogenic lineages.The advances that we hope to make with this project will not only serve as a basis for more detailed investigation of the links between the cell microenvironment and gene expression, but will also enable strategies to be developed to control cell behaviour in tissue engineering and therapeutic applications.