(229q) Cytoplasmic Stiffness in Migrating Cells at the Interface of a Chemical/Mechanical Gradient

In order to migrate, cells must exhibit a certain degree of anisotropy resulting in a well-defined leading and trailing edge. Cell polarization and the redistribution of cytoskeletal components are key steps in cell migration. Therefore, we seek to investigate how substrate rigidity and chemical composition could affect cellular cytoplasmic stiffness. Our goal is to correlate anisotropy in cytoplasmic stiffness to migratory speed and directions. Such studies will provide fundamental insights into wound healing, cancer cell metastasis and in developmental biology. We have investigated the changes in cytoplasmic stiffness of fibroblasts on chemical-only, mechanical-only and dual chemical-mechanical gradient substrates developed previously in our group¹. These studies allow us to make correlations between substrate rigidity, protein concentrations, cytoplasmic elasticity, and migration.

Polyacrylamide hydrogels exhibiting chemical, mechanical and opposing dual mechanical/chemical gradients were designed according to previously established methods¹. Cytoplasmic stiffness measurements were conducted on BALB/c 3T3 fibroblasts using a Veeco Bioscope II AFM equipped with a heated stage. The AFM was configured with a Nikon TE-2000U optical microscope allowing for precise positioning of the AFM cantilever over the cellular region of interest, as well as conducting fluorescence microscopy of cytoskeletal components. Force distance curves were collected from different regions such as nuclear, peri-nuclear and lamellipodia, leading and trailing edges of the cell. Correlations to cell cytoplasmic stiffness and the organization of the actin cytoskeleton will be made by imaging fibroblasts labeled with CellLight® Actin-GFP (Life Technologies) through confocal microscopy immediately preceding AFM measurements.

For our preliminary studies, AFM measurements have been taken on fibroblasts seeded on collagen-coated glass coverslips or polyacrylamide (PAAM) hydrogels. The Youngâ??s modulus (YM) of the PAAM gels was either 45 or 120kPa. The elastic modulus was obtained from fitting force- distance curves with the Sneddonâ??s modified Hertz model. Cells were indented up to 1μm and the resulting force distance curves were fit for indentations up to 10% of the cellâ??s thickness to avoid substrate effects. Force distance curves obtained from single cells were collected within a minuteâ??s timespan to minimize interactions between the cell and AFM cantilever. We have observed YM ranging from 1-20kPa with as much as a 12-fold increase in cytoplasmic stiffness between the perinuclear and peripheral regions of fibroblasts seeded on rigid glass coverslips. When fibroblasts were cultured either on the 45 or 120kPa PAAM gels, similar trends were observed. On the 45kPa gels the decrease in YM between the stiff periphery and soft perinuclear regions was approximately 2-fold. However, on the 120kPa gels, the corresponding decrease was approximately 6-fold. These trends demonstrate that the substrate rigidity modulates the cytoplasmic stiffness of cells. More importantly, substrate rigidity appears to play an important role in the anisotropy of cellular cytoplasmic stiffness.

We will correlate anisotropies in cytoplasmic stiffness to cytoskeletal and focal adhesion protein expression. The use of controlled gradient substrates enables us to separate multiple influences to cellular locomotion. These studies will allow us to gain a greater understanding of the complex interplay between the cytoskeleton, substrate properties and migratory processes.

References: 1.) Jain G, ACS Biomater. Sci. Eng., 2016; 8:621-631. 2.) Solon J, Levental I, Biophys. J., 2007;93:4453-61.