Significance: Cell volume is thought to be a well-controlled cellular characteristic, increasing as a cell grows, while macromolecular density is maintained. We report that cell volume can also change in response to external physical cues, leading to water influx/efflux, which causes significant changes in subcellular macromolecular density. This is observed when cells spread out on a substrate: Cells reduce their volume and increase their molecular crowding due to an accompanying water efflux. Exploring this phenomenon further, we removed water from mesenchymal stem cells through osmotic pressure and found this was sufficient to alter their differentiation pathway. Based on these results, we suggest cells chart different differentiation and behavioral pathways by sensing/altering their cytoplasmic volume and density through changes in water influx/efflux.

Significance: The behavior of cells is strongly affected by the mechanics of their surroundings. In tissues, cells interact with the extracellular matrix, a 3D network of biopolymers with a highly nonlinear elastic response. We introduce a method exploiting this matrix nonlinearity to infer mechanical stresses in 3D. Using this method, we demonstrate that cell contractility induces large stresses, which generate a massive stiffness gradient over an extended region in 3D matrices of collagen, fibrin, and Matrigel. Our work highlights the importance of nonlinear matrix mechanics at the microscopic scale and suggests a concrete mechanism through which cells can control their microenvironment and mechanically communicate with each other.

Significance: Intermediate filaments (IFs) remain the least understood with respect to their functions in mammalian cells even though they have been related to many devastating human diseases. Here we use optical tweezers to perform micromechanical measurements in living cells and in IF enriched cytoskeletons devoid of actin and microtubules. We identify that cytoskeletal vimentin IFs (VIFs) provide cells with a hyperelastic rubber-like network that regulates the essential mechanical properties of mammalian cells including stretchability, strength, resilience, and toughness. We show that VIFs maintain cell integrity and viability under conditions involving extreme deformations. We further show that the stretchy VIF network can effectively disperse locally induced mechanical stress to larger regions within individual cells, enabling the dissipation of energy throughout a cell.

Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V. We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s⁻¹ < V/a < 2 s⁻¹) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s⁻¹ < V/a < 80 s⁻¹). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales.