We performed a 40 ns simulation of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18(3)) in a 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) bilayer in order to facilitate interpretation of lipid dynamics and membrane structure from fluorescence lifetime, anisotropy, and fluorescence correlations spectroscopy (FCS). Incorporation of DiI of 1.6 to 3.2 mol% induced negligible changes in area per lipid but detectable increases in bilayer thickness, each of which are indicators of membrane structural perturbation. The DiI chromophore angle was 77 +/- 17 degrees with respect to the bilayer normal, consistent with rotational diffusion inferred from polarization studies. The DiI headgroup was located 0.63 nm below the lipid head group-water interface, a novel result in contrast to some popular cartoon representations of DiI but consistent with DiI's increase in quantum yield when incorporated into lipid bilayers. Importantly, the fast component of rotational anisotropy matched published experimental results demonstrating that sufficient free volume exists at the sub-interfacial region to support fast rotations. Simulations with non-charged DiI head groups exhibited DiI flip-flop, demonstrating that the positively-charged chromophore stabilizes the orientation and location of DiI in a single monolayer. DiI induced detectable changes in interfacial properties of water ordering, electrostatic potential, and changes in P-N vector orientation of DPPC lipids. The diffusion coefficient of DiI (9.7 +/- 0.02 x 10(-8) cm2 s(-1)) was similar to the diffusion of DPPC molecules (10.7 +/- 0.04 x 10(-8) cm2 s(-1)), supporting the conclusion that DiI dynamics reflect lipid dynamics. These results provide the first atomistic level insight into DiI dynamics, results essential in elucidating lipid dynamics through single molecule fluorescence studies.

Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from −2.6 mN m−1 to 15.9 mN m−1. Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing.

Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.

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.

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