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Bone is a structure finely tuned to its mechanical environment. Strain detection in the skeleton is thought to be effected by bone lining cells and osteocytes embedded within the bone matrix. However, because bone is a composite material with multiple enclosed cavities and a complex architecture, the strain to which cells are subjected to in vivo and to which they react remains unknown. This thesis examined the sensitivity of cells of the osteoblastic lineage to mechanical strain using three different methods. First, Atomic Force Microscopy (AFM) was used to mechanically stimulate the cells and measure their elasticity, whilst changes in intracellular calcium concentrations were monitored with a confocal microscope. Post-hoc, the strain applied was calculated. The proportion of cells reacting by increasing their intracellular calcium concentrations followed a dose response curve with 50% of the cells reacting for 2.5% strain. Second, patch-clamp electrophysiology and simultaneous video-microscopy were combined to provide an experimental curve relating the length of the membrane extension into the micropipette to the aspiration pressure and the opening of mechanosensitive channels in response to pressure. A finite element simulation of the membrane aspiration into the micropipette revealed that strains superior to 20% were needed to open mechano-sensitive channels. Third, digital strain estimation was applied to Green Fluorescent Protein-actin or tubulin transfected melanoma cells to provide an estimate of the strain magnitude needed to elicit a rise in intracellular calcium concentration in response to micropipette poking. Intercellular communication was also examined. Many studies in vitro have studied the mechanisms of cellular adaptation to mechanical strains; however, the applied strains remain unknown and results from different techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by AFM with finite element modelling, we calculated the cellular strains exerted by common whole cell straining techniques and micromanipulation techniques.

Cell competition is a phenomenon in which less fit cells are removed from a tissue for optimal survival of the host. Competition has been observed in many physiological and pathophysiological conditions, especially in the prevention of tumor development. While there have been extensive population-scale experimental studies of competition, the competitive strategies and their underlying mechanisms in single cells are poorly understood. To date, two main mechanisms of cell competition have been described. Mechanical competition arises when the two competing cell types have different sensitivities to crowding. In contrast, during biochemical competition, signaling occurs at the interface between cell types leading to apoptosis of the loser cells. However, rigorously testing these hypotheses remains challenging due to the difficulty of obtaining sufficient single cell level information to bridge scales to the whole tissue. In this thesis, I present metrics aimed at characterising competition at the single cell level. Then, I demonstrate the development of a multi-layered, cell-scale computational model that I use to gain understanding on the single cell mechanisms that govern mechanical competition and decipher the "rules of the cellular game". After benchmarking cell growth and homeostasis in pure populations, I show that competition emerges when both cell types are included in simulations. I then investigate the impact of each computational parameter on the outcome of cell competition. Intriguingly, the outcome of biochemical competition is controlled by topological entropy between cell types, whereas the outcome of mechanical cell competition is exclusively controlled by differences in energetic potential between cell types. As 90% of cancers arise from epithelia and a number of genetic diseases present symptoms of epithelial fragility, I anticipate that my model of realistic implementation of epithelia will be of use to the biophysics and computational modelling community.

Cells actively regulate their lipid composition and localisation during cell division, with both signalling roles and structural roles likely. In this study, I investigate the specific roles of lipids in the formation and maintenance of cell-cell junctions, and in cell migration. Although massive membrane rearrangements occur during both processes and lipids are fundamental building blocks of cell membranes, the roles of lipids remain unclear. After perturbing lipid metabolism enzymes with an siRNA library targeting 260 lipid biosynthetic enzymes in HaCaT (immortalized human keratinocytes), junctions were visualised using the adherens junction (AJ) marker α-catenin, and the resultant junction morphologies were examined. The primary screen revealed a potential role for 16 enzymes with two distinct junctional phenotypes: intercellular gaps and abnormal junction morphology. A secondary migration screen based on wound healing assays was conducted with a subgroup of these hits. I found that depletion of some enzymes results in defects in migration speed and cohesiveness, which suggests potential important roles for those enzymes and the lipids they make in cell migration. AGPAT2 (1-Acylglycerol-3-Phosphate O-Acyltransferase 2) is one of the hits being prioritised for further experiments to understand the role of related lipids. The depletion of AGPAT2 changes cell-cell junction morphology dramatically. In HaCaT, AJs expand on the neighbouring cells, while in Caco-2 (human colorectal adenocarcinoma cells), tight junctions (TJs) show an undulating morphology. Lipidomic analysis conducted following AGPAT2 depletion identified increases in triacylglycerols (TAGs), decreases in ether phosphatidylcholines (PCs) and reduced membrane fluidity indices. Further experiments are being performed to better characterise the phenotypes and identify the exact roles of the lipids. Overall, my study provides evidence that lipids may be involved in cell migration and cell-cell junction formation and maintenance and suggests that proper lipid metabolism is essential for regulating cell-cell junction and migration.

Epithelial monolayers are a class of animal tissue which comprise some of the most basic and important structures in metazoans. Many remarkable morphogenetic events, responsible for determining adult tissue shape, take place in epithelia and they continue to perform crucial functions throughout adult life. Whether it is the filling of the bladder or during a precise morphogenetic transformation, epithelia must frequently undergo or drive tissue deformations. Therefore, much effort has been directed towards understanding the combination of material properties and cellular behaviours which determine how epithelia respond to the application of stress and strain. The complex biophysical environment of in vivo tissues, however, can hinder attempts to understand the underlying mechanisms and principles at play. To address this, a novel and highly simplified system is utilised in which uniaxial strain is applied to epithelia monolayers which are devoid of a substrate. Application of compressive strain to these suspended epithelia unexpectedly revealed their ability to autonomously flatten buckles and remodel cell shape as the tissue mechanically adapts to a new shorter length over a duration of ~60 seconds. These changes, which are found to be driven by actomyosin contractility, appear fully reversible since the epithelia can readapt to their initial length when it is restored and maintained over a similar time period. At longer timescales, cell division within the epithelia is also found to be affected by the application of strain. Both compressive and tensile strain causes an alignment of division orientation which is demonstrated to be due to a global realignment of cell long axes combined with orientation of division along these axes, rather than by cells detecting and responding to long-range tissue stress orientation. In turn, these strain-oriented cell divisions homeostatically alter tissue organisation by redistributing cell mass along the direction of division and ultimately restore isotropic cell shape.

As interest in biophysics and biophysical modelling has grown in the cell and developmental biology communities, a variety of techniques have been developed to measure the mechanical properties of single cells. Atomic Force Microscopy (AFM) has become one of the preferred methods for these measurements primarily due to its ease of operation and commercial availability. However, measurements on soft cells with a variable surface topography require an additional level of care so that the predicted contact area with the cell surface is accurately estimated. Using combined AFM and confocal microscopy I have shown that with pyramidal tipped cantilevers the cell body can easily deform to the shape of the tip but can also touch the underside of the AFM cantilever beam causing an overestimation of elasticity. Such artefactual increases in contact area could be avoided by using spherical tipped cantilevers or tips with a high aspect ratio. I examined the role of the cytoskeleton and cell contractility in setting single cell stiffness with AFM. With techniques such as AFM, the rheology of single cells is becoming increasingly well characterised. The next logical step in furthering our understanding of organ and embryo mechanics is to scale up investigations to simple tissues such as on cell thick monolayers. I have developed methods to measure the mechanical properties of MDCK epithelial cell monolayers under AFM indentation or planar extension. Using deep indentation of monolayers cultured on soft gels I have measured the evolution of mechanical properties upon the establishment of cell-cell junctions. The relative mechanical stiffnesses of monolayer-gel composites evolve as cell contacts are established and required the formation of mature contractile adherens junctions. To measure the planar mechanical properties of cell monolayers I designed a system to create monolayers freely suspended from their susbstrate between two test rods. Cell monolayers have a higher stiffness than their cellular constituents due to the organisation of the cell cytoskeleton upon the formation of matured intercellular junctions.