Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix

Abstract

Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

Optical measurement of arterial mechanical properties: from atherosclerotic plaque initiation to rupture

“…In contrast, in optical microrheology, the mean square displacement (MSD) of tracer probes or endogenous particles is quantified using video microscopy or dynamic light scattering techniques, and the estimated MSD is related with the complex frequency-dependent viscoelastic modulus, G*, via the generalized Stokes-Einstein relation. [61][62][63] Over the past decade, microrheology approaches have been extended to biological systems to measure the viscoelastic moduli of living cells, subcellular components, and ECM proteins using synthetic [64][65][66] or endogenous light scattering particles 66 as well as fluorescently labeled nano-and microbeads as tracer probes. 67,68 In particle tracking microrheology, micron-sized fluorescent beads are injected within a living cell and passive bead displacements are subsequently tracked using a fluorescence microscope and a high-frame-rate camera to measure the mechanical compliance of the cytoplasm over time scales of 0.1 to 10 s (Fig.…”

Why should polymer physicists study biopolymers?

“…Microrheology, developed for the study of complex fluids, has been extensively applied to solutions of the cytoskeletal polymer actin 32 and more recently still to cell contents. [33][34][35] Theoretical ideas on entangled semiflexible polymers can be applied to analyze the anticipated behavior of the cytoskeleton under different conditions, 36 and coupled with microrheological measurements to compare with real cells. When cells adhere to surfaces they produce a complex glue of polymers known as the extracellular matrix.…”

Linking particle dynamics to intracellular micromechanics in living cells

“…Alternatively, probes with selectable size and surface chemistry have also been injected into cells either by a ballistic gun or a microinjector (70)(71)(72)(73)(74). In certain cells, microinjected and ballistically injected particles (BIPs) have been found to behave similarly (70).…”

“…In several papers (70)(71)(72)(73)(74), IPT had been presented as a microrheological method, allowing the measurement of viscoelastic moduli of the cell interior, at the timescales (typically 0.1-30 s) accessible to video microscopy. However, other papers had demonstrated violations of the fluctuation-dissipation theorem that underlies the GSER used in IPT.…”