Joanna L. MacKay

Probing cellular mechanobiology in three-dimensional culture with collagen-agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

A Genetic Strategy for the Dynamic and Graded Control of Cell Mechanics, Motility, and Matrix Remodeling

Cellular mechanical properties have emerged as central regulators of many critical cell behaviors, including proliferation, motility, and differentiation. Although investigators have developed numerous techniques to influence these properties indirectly by engineering the extracellular matrix (ECM), relatively few tools are available to directly engineer the cells themselves. Here we present a genetic strategy for obtaining graded, dynamic control over cellular mechanical properties by regulating the expression of mutant mechanotransductive proteins from a single copy of a gene placed under a repressible promoter. With the use of constitutively active mutants of RhoA GTPase and myosin light chain kinase, we show that varying the expression level of either protein produces graded changes in stress fiber assembly, traction force generation, cellular stiffness, and migration speed. Using this approach, we demonstrate that soft ECMs render cells maximally sensitive to changes in RhoA activity, and that by modulating the ability of cells to engage and contract soft ECMs, we can dynamically control cell spreading, migration, and matrix remodeling. Thus, in addition to providing quantitative relationships between mechanotransductive signaling, cellular mechanical properties, and dynamic cell behaviors, this strategy enables us to control the physical interactions between cells and the ECM and thereby dictate how cells respond to matrix properties.

Measuring the Elastic Properties of Living Cells with Atomic Force Microscopy Indentation

Atomic force microscopy (AFM) is a powerful and versatile tool for probing the mechanical properties of biological samples. This chapter describes the procedures for using AFM indentation to measure the elastic moduli of living cells. We include step-by-step instructions for cantilever calibration and data acquisition using a combined AFM/optical microscope system, as well as a detailed protocol for data analysis. Our protocol is written specifically for the BioScope™ Catalyst™ AFM system (Bruker AXS Inc.); however, most of the general concepts can be readily translated to other commercial systems.

A fluorescent peroxidase probe increases the sensitivity of commercial ELISAs by two orders of magnitude.

The low detection sensitivity of enzyme linked immunosorbent assays (ELISAs) is a central problem in science and limits progress in multiple areas of biology and medicine. In this report we demonstrate that the hydrocyanines, a family of fluorescent reactive oxygen species (ROS) probes, can act as turn on fluorescent horseradish peroxidase (HRP) probes and thereby increase the sensitivity of conventional ELISAs by two orders of magnitude.

Constitutive Activation of Myosin-Dependent Contractility Sensitizes Glioma Tumor-Initiating Cells to Mechanical Inputs and Reduces Tissue Invasion

Glioblastoma (GBM) tumors are thought to arise from a subpopulation of brain tumor-initiating cells (BTICs), which mediate therapeutic resistance and seed new tumors. Virtually nothing is known about the role of extracellular matrix (ECM) mechanical inputs in controlling BTIC invasion and tumorigenesis, despite the recognized importance of these signals in tissue dysplasia and cell motility. Here we show that human BTICs can evade the inhibition of spreading, migration, and proliferation normally imposed by compliant ECMs. Remarkably, activation of myosin II-dependent contractility restores this inhibition, strongly restricts BTIC ECM invasion in vitro, and dramatically extends survival and limits infiltration in vivo. This is the first direct evidence that manipulation of mechanotransductive signaling can alter the tumor-initiating capacity of brain TICs, supporting further exploration of these signals as potential therapeutic targets.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Tuning Cellular Mechanics and Motility through Genetic Manipulation of Rho GTPase and Myosin Activity

The mechanical properties of cells such as size, shape, and stiffness have recently been recognized as important regulators of cell behavior. For example, cells cultured on small areas of extracellular matrix (ECM) grow slower than cells on large areas (Chen et al., Science 1997), and cells confined to one-dimensional lines elongate their bodies and migrate faster than cells cultured on two-dimensional substrates (Doyle et al., JCB 2009). While there are many strategies to indirectly control such behaviors through manipulation of the ECM, there is a marked lack of strategies for controlling these behaviors in more direct ways. To address this need, we have developed a genetic strategy for directly tuning the mechanical properties of cells. Specifically, we have placed genetic mutants of mechanotransductive proteins, including RhoA and myosin light chain kinase (MLCK), under a conditional promoter and introduced a single copy into glioblastoma cells using viral vectors. By expressing these proteins from a conditional promoter, we can vary their expression by simply changing the inducer concentration in the culture medium. RhoA and MLCK are known to activate myosin II, which is the motor protein responsible for force generation, and with constitutively active (CA) mutants of these proteins, we can modulate cytoskeletal architecture, force generation, and cellular stiffness in a graded fashion. Moreover, by switching expression of these proteins on and off, we can dynamically control cell spreading, migration, and ECM remodeling. We believe this strategy will serve as a valuable tool for developing quantitative relationships between intracellular signaling, cellular mechanics, and complex behaviors. Furthermore, such precise control over cell behavior could allow us to dictate how cells physically interact with their microenvironment, which would be particularly useful in tissue engineering applications that interface cells with synthetic materials.