Mechanics of the cellular microenvironment as perceived by cellsin vivo

Abstract

Tissue morphogenesis and repair, as well as organ homeostasis, require cells to constantly monitor their 3D microenvironment and adapt their behaviors in response to local biochemical and mechanical cues1-6. In vitro studies have shown that substrate stiffness and stress relaxation are important mechanical parameters in the control of cell proliferation and differentiation, stem cell maintenance, cell migration 7-11, as well as tumor progression and metastasis12,13. Yet, the mechanical parameters of the microenvironment that cells perceive in vivo, within 3D tissues, remain unknown. In complex materials with strain- and time-dependent material properties, the perceived mechanical parameters depend both on the strain and timescales at which the material is mechanically probed14. Here, we quantify in vivo and in situ the mechanics of the cellular microenvironment that cells probe during vertebrate presomitic mesoderm (PSM) specification. By analyzing the magnitude and dynamics of endogenous, cell-generated strains, we show that individual cells preferentially probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. We reveal how stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. While stress relaxation timescales are spatially uniform in the tissue, most mechanical parameters, including those probed by cells, vary along the anteroposterior axis, as mesodermal progenitors commit to different lineages. Understanding the mechanical parameters that cells probe in their native 3D environment is important for quantitative studies of mechanosensation in vivo2-4,6,15 and can help design scaffolds for tissue engineering applications16-18.

Fluorous Soluble Cyanine Dyes for Visualizing Perfluorocarbons in Living Systems

“…Overall, all of the fluorous cyanine dyes display greater solubility in perfluoromethyl cyclohexane than does fluorous rhodamine 3, the current standard for tracking the fluorous phase in biological environments. 24,25 While solubility in perfluorocarbons was our primary interest, we also evaluated the solubility of the cyanine fluorofluorophores in a range of solvents (Figures S4-S7). We found that the fluorous cyanine dyes were well-solubilized in acetone and ethanol.…”

“…These solvents were selected due to their use as a standard in the fluorous community (PFMC), 2 their prominence as the inner phase of perfluorocarbon nanoemulsions (PFOB and PFD), 8 and their applications toward in vivo force measurements (HFE-7300 and HFE-7700). 25,32 Upon visual inspection, the fluorous-tagged cyanine dyes appeared soluble in all of these solvents. Yet, the absorption spectra revealed that, with the exception of PFOB, F 86 Cy7 (1c) was aggregated in solution (Figure S6).…”

“…We have capitalized on the inert, orthogonal nature of the fluorous phase to engineer a perfluorocarbon (PFC) microdroplet technique to precisely measure endogenous mechanical stresses and spatial variations in tissue material properties in vivo and in situ. 10,24,25 These techniques allow quantitative mechanobiology studies in the native environment of cells using confocal microscopy.…”

“…Rhodamine 3 is currently employed for visualizing the fluorous phase in living systems. 24,25 Finally, we showcase the utility of cyanine 1b for multiplexed microscopy of perfluorocarbon nanoemulsions in cells and for force measurements in 3D spheroids and zebrafish embryonic tissues using perfluorocarbon microdroplets.…”

Viscoelastic Networks: Forming Cells and Tissues

“…However, whether such measurements are relevant to the time window of morphogenesis still needs to be addressed, since such measurements show how a tissue deforms when applying an exogenous force for a certain time. How these features compare to the magnitude and duration of the endogenously exerted forces is still unclear [9,22,23]. In addition, during such experimental measurements, the applied force typically triggers a large deformation of the material (nonlinear regime).…”