Physiological Membrane Tension Causes An Increase In Lipid Diffusion: A Single Molecule Fluorescence Study

Abstract

Human mesenchymal stem cells (hMSCs) are mechanosensitive and specify their lineage based on the stiffness of their environment. That is, a purely mechanical cue is sufficient to cause cells on gels with an intermediate stiffness (Young's modulus E ¼ 11 kPa) to adopt a spindle-like morphology characteristic of muscle cells and to up-regulate muscle markers such as MyoD. Inhibition of myosin II by blebistatin blocks this lineage specification. We analyzed the shape changes that occur at early times in cells cultured on soft to stiff (1, 11, and 34 kPa) gels using an automated image analysis algorithm to correlate morphological changes with stress fiber formation and orientation. While the total production of stress fibers increases monotonically with substrate stiffness similar to the trend in projected cell area, the orientation shows a maximum at intermediate stiffness, similar to the polarization. These early time changes are not due to changes in gene expression, which occur only after several days, but must instead have a more rapid biophysical basis. Both the myosin inhibition and correlation between stress fiber orientation and cell morphology suggest a critical role for these contractile structures in mechanosensing. To help dissect their function we used a multi-color hybrid fluorescence and atomic force microscope to locate specific stress fiber proteins such as actin and myosin and to correlate their presence with specific features in the high resolution AFM images. AFM also offers the possibility of determining the mechanics of the stress fibers themselves, and thus of integrating their properties into a more complete picture of the cell's mechanics and ultimately mechanosensitivity.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes

“…For example, raising the membrane tension from 0 to 15 mN/m resulted in a 52% increase in the diffusion coefficient . This behavior was also reported in GUV experiments, where a 44% faster diffusion was found for an increase in membrane tension from 0.02 to 0.1 mN/m …”

“…25 This behavior was also reported in GUV experiments, where a 44% faster diffusion was found for an increase in membrane tension from 0.02 to 0.1 mN/m. 27 In addition, a curvature-induced effect might play a role. Considering the large coverage of the GNPs with streptavidin, it is possible that more than one lipid is bound to a GNP.…”

Phase separation of biomimetic membranes:

“…This dependency of membrane tension on diffusion was also shown in molecular dynamics simulations (MDs) [256,257] and was also measured using FCS on GUVs, using micro pipette aspiration to tune the GUV tension. [258] Interestingly, bulk methods like fluorescence recovery after photobleaching (FRAP) on GUVs and the micro pipette aspiration did not enable to measure the increase found in single lipid diffusion studies. [259] The GNPs on the s-PSM with the p-Si 3 N 4 functionalization were slower than the GNPs on the s-PSM with the 6MH-Au functionalization (figure 5.9 B, table 5.2).…”

“…[170,[264][265][266] However, as stated before this can be explained by the increased membrane tension of f-PSM compared to GUVs. [256][257][258] The membrane tension of GUVs can be assumed to be zero and the membrane tension of the f-PSM composed of DOPC on SiO x functionalized substrates was 10.5 mN m −1 (table 5.1).…”