Myosin II Activity Softens Cells in Suspension

Abstract

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension.To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

Mapping intracellular mechanics on micropatterned substrates

“…S4) and intermediate filaments in the perinuclear region (59,62). There has recently been a growing interest in the role of active cellular forces, such as forces generated by cytoskeletal dynamics, molecular motors, or acto-myosin contractility, in intracellular mechanics, and conflicting results have been reported (8,41,44,(63)(64)(65). Here, we measure an unambiguous stiffening of the cytoplasm of RPE1 cells upon energy depletion, reflected by an elevated shear modulus localized at the cell center up to 5 μm away from the nucleus (Fig.…”

“…Using PL rheology, we analyze the relaxation of internalized microspheres following an imposed displacement in an optical trap and measure the local intracellular complex shear modulus G. The real part of G, the storage modulus G′, is related to the elasticity of the bead microenvironment, whereas the imaginary part of G, the loss modulus G″, is related to its viscous-like behavior. We also use the more classical standard linear liquid (SLL) viscoelastic model (19,41) to analyze the relaxation experiments in terms of viscoelastic components and compare the results with our PL-based model. The resulting maps characterize the spatial variations of the intracellular Significance Cell mechanics is crucial for many, if not all, cell functions.…”

Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device

“…For proof-of-principle studies, we focused on NIH 3T3 fibroblasts, which have been mechanically characterized by AFM (46,47), optical stretching (48,49), and MPA (50). To explore the potential of our cross-slot device for novel discovery, we also characterized GBM TICs, a stem-like subpopulation of GBM tumors thought to drive tumor initiation, recurrence, and therapeutic resistance (26,51,52).…”

Physical probing of cells

“…Distributions of optical deformability were so broad, however, that unambiguous classification of single cells was not possible [41]. Chan et al recently performed measurements on a variety of physiologically adherent and non-adherent cells and also perturbed cell mechanics using biochemical drugs such as blebbistatin, a potent and selective inhibitor of the ATPase of myosin II's, thereby weakening acto-myosin contractility [42]. Interestingly, cells became effectively stiffer with blebbistatin treatment, which is in contrast to measurements by AFM and the dual optical trap [43].…”