Physical regulation of cell surface access and cell adhesion by membrane-anchored hyaluronan
Chang, Patrick S.
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Pericellular matrix (PCM) is a hyaluronan rich polymer matrix anchored to the outer cell membrane surface. Negatively charged bottlebrush proteoglycans also play an important role in the matrix giving it its structure. In the past decade, it has been increasingly appreciated that this often microns-thick cell coat is involved in regulating a range of key physiological processes, including proliferation, migration, synaptogenesis, and wound healing. How the PCM impacts these processes is not well understood, but it is likely in part via physical and/or physiochemical processes, resulting from the bulky size and high negative charge of hyaluronan and its associated proteoglycans. This thesis work introduces new methods to quantify and probe the physical function of the PCM, and addresses two fundamental biophysical questions (i) how the PCM impacts transport of objects to the surface and (ii) whether surface-associated hyaluronan matrix mechanically regulates cell adhesion. A detailed picture of the in vitro PCM microstructure and its impact on the transport of molecules and particles to the cell surface is reported for the first time. The work shows that particles 40 nm and larger are significantly impacted by the PCM, which acts to filter and reduce the number reaching the surface in a size-dependent fashion – a consequence of the decreasing pore size of the PCM towards the cell surface. Molecular accessibility to the cell is less impacted unless the species is positively charged, in which case studies show that it sequesters strongly within the PCM, binding to the negatively-charged bottlebrush proteoglycans. Studies with exogenous proteoglycans demonstrate opportunities to modify the thickness and pore size of the sieve-like PCM. Addition of proteoglycan into the matrix extends the matrix’s thickness and decreases its pore size. Different concentrations of exogenous proteoglycan provide the ability to tune the matrix reach and tightness. Mechanical quantification of cell adhesion strength versus hyaluronan patches at the cell interface reveals that PCM reduces adhesion to substrates in several cell types. Complementary studies with interferometric microscopy reveal that HA increases the roughness of the cell interface at the substrate, reducing contact, potentially weakening adhesion. The outcome of these detailed studies strongly imply that the role of cell-surface associated hyaluronan in vivo deserves much more attention than previously garnered. The PCM acts as a gatekeeper in modifying what reaches and adheres to the cell surface – two processes fundamental in the regulation of multicellular organisms.