Compressive Mechanics of Hyaluronan Brushes - A Study with a Combined Colloidal Probe AFM/RICM Setup

Abstract

Around many biological cells, in the transition zone between the plasma membrane and the extracellular matrix, lies the micrometer thick pericellular coat (PCC): hyaluronan (HA), a long and flexible, charged glycosaminoglycan and HA-binding proteins self-organize into a hydrated gel-like coat which serves crucial mechanical functions of the cell. For a thorough investigation of the physical principles underlying the biological functions of these coats and provided the instrumental limitations in studying these highly hydrated systems in vivo, model systems are useful.Atomic force microscopy (AFM) is a widely used analytical approach to determine the behavior of molecules or thin films under mechanical force. Colloidal probe reflection interference contrast microscopy (RICM) is an established microinterferometric technique to determine the thickness of soft hydrated films. In this study we combine colloidal probe AFM and RICM to investigate the mechanical properties of a well-defined model system of the pericellular coat: films of hyaluronan that is grafted to supported lipid bilayers (Richter et.al. 2007, JACS, 129:5306-7). The combination provides interaction forces as a function of the absolute distance between the two approaching surfaces, information that cannot easily be obtained with either technique alone. We quantify the thickness of HA films, and their resistance to compression forces as a function of external salt concentration. From the experimental data, and comparison with scaling and mean-field theories, we conclude that grafted HA films are well-described as a polyelectrolyte brush. Addition of cartilage proteoglycan aggrecan induced a drastic increase in thickness and resistance to compression. The novel combined AFM/RICM setup can serve as a powerful tool to quantify the mechanical properties of soft hydrated biopolymer films with precise control of probe sample separation.