Stem cell mechanoadaptation. II. Microtubule stabilization and substrate compliance effects on cytoskeletal remodeling.

Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate as well as the emergence of tissue structure and function. Here, in the second part of a two-part experimental series, we aimed to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells and their nuclei. Akin to mechanical testing of the most basic living and adapting unit of life, i.e., the cell, in model tissue templates, we probed native and microtubule-stabilized (via Paclitaxel, PAX, exposure) stem cells' cytoskeletal adaptation capacity on substrates of increasing compliance (exerting local tension on cells) and with increased target seeding densities (exerting local compression on cells). On 10 and 100 kPa gels, cells seeded at 5000 cells/cm and cells proliferated to 15 000 cells/cm exhibited bulk moduli that nearly matched those of their respective substrates; hence, they exhibited a greater increase in Young's Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced microtubule-stabilized cells' F-actin, and microtubule concentration increases compared to cells seeded on glass. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which, in turn, exerts a larger effect in determining cells' capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells. Hence, the studies progress our understanding of the dynamic cytoskeleton as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing.