Cell mechanics and cell-cell interactions of fibroblasts from Dupuytren's Patient : Atomic Force Microscopy Investigation

Abstract

Cells as a biological entity of tissue, itself made of biomolecules such as mostly proteins, lipids and carbohydrates, creates its own meshwork of biopolymers named extracellular matrix (ECM) particularly fibroblasts. With the advanced light and force microscopies, inter-cellular, cell-ECM and intracellular signaling pathways are deeply explored either by tagging the biomolecule of interest with fluorophores or by applying certain forces(in the order of pN to nN). In the field of mechanobiology, interplay between cell function and physical forces are studied using biophysical tools thatprobe their diverse mechanisms. Cells exert forces("inside-out" signalling)and also respond to physical forces from their micro-environment("outside-in" signalling) through participation of chain of varying protein signaling molecules. Actin molecules from cytoskeleton family form filaments in the cytoplasmic side of the cell and myosin walk on these filaments generates contractile tension. These traction forces get transmitted to the extracellular matrixof the cell or to the neighboring cells through protein complexes such as integrin and cadherins, respectively. Fibroblasts,from the mesenchymal family, are the abundant cells found in the connective tissue. Basically, fibroblasts synthesize,degrade and maintain the extracellular matrix components of the tissue. Fibroblasts, by acquiring different phenotypes called protomyofibroblast/myofibroblast, play a huge participation in various connective tissue related diseases. Myofibroblast are large cells possessing large bundles of actin filaments of isomers named alpha smooth muscle actin (I /--SMA). On the other hand, protomyofibroblast share the similar characteristic appearance but shows I /--SMA negative large stress fibres. In Dupuytrena s disease, thesemyofibroblasts persists and deform the surrounding matrix environment thus results in tissue stiffening and further leads to tissue contracture. Existing various biophysical tools maps forces such as tractile force, cell-cell interaction force and cell-ECM interaction force. One among such tool is Atomic Force Microscopy, a multifunctional toolbox in cellular biology to observe various cell types mechanics. Observing cell viscoelastic properties by application of controlled force (nanonewton) to the adherent cell become more common in the biomedical community. This thesis demonstrates the measurement of viscoelastic properties of fibroblast of different phenotypes extracted from a Dupuytrena s diseased patient and ECM derived from various tissues.The bio-mechanical interplay between cell and ECM has been studied with careful design of the AFM experiments. Fibroblasts extracted from the cords and nodular region of the palmar fascia exhibits myofibroblast phenotype and migrate slower than the fibroblast extracted from dermal and scar region. Normal and scar fibroblasts migrate faster in the wound healing assay.On the decellularized matrices, scar fibroblasts exhibitprotomyofibroblast phenotype by expressing large stress fibres. Whereas, normal fibroblasts derived from the dermal region express the healthy phenotypic appearance. From AFM based Single-cell force spectroscopy (SCFS), cell-cell interaction force measurements evaluatethe homophilic and heterophilic cadherinpairs mechanical bond strength expressed in homo-cellular (fibroblast of similar phenotype) and hetero-cellular (fibroblast-epithelial cell) arrangements. SCFS measurements also illustrate the significant role of actomyosin contractile apparatus in cadherin extracellular iidomain binding dynamics. With this evidence, SCFS setup has become an excellent spectroscopic tool to study the intracellular signalingcascades that are linked to the extracellular domain consisting transmembrane proteins such as cadherins. Therefore, an understanding of the unique fibroblasts mechanobiology is necessary to study the healthy and diseased tissue dynamics. The cell-cell and cell-ECM bio-chemical and bio-mechanical cues are strongly interdependent. Finally, the current thesis opens the basic understanding of the fibroblasts biophysical properties using AFM nano-mechanical tool and unravels the fibroblasts biomechanical function in sub-tissue level biology.