Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. The objectives of this study were to develop a calcium phosphate nanoparticle encapsulation system for organic dyes and therapeutic drugs so that advanced fluoresence methods could be used to assess the efficiency of drug delivery and possible mechanisms of nanoparticle bioabsorption. Highly concentrated CPNPs encapsulating a variety of organic fluorophores were successfully synthesized. Well-dispersed CPNPs encapsulating Cy3 amidite exhibited nearly a 5-fold increase in fluorescence quantum yield when compared to the free dye in PBS. FCS diffusion data and cell staining were used to show pH-dependent dissolution of the particles and cellular uptake, respectively. Furthermore, an experimental hydrophobic cell growth inhibitor, ceramide, was successfully delivered in vitro to human vascular smooth muscle cells via encapsulation in CPNPs. These studies demonstrate that CPNPs are effective carriers of dyes and drugs for bioimaging and, potentially, for therapeutic intervention.

Blood flow-associated shear stress causes physiological and pathophysiological biochemical processes in endothelial cells that may be initiated by alterations in plasma membrane lipid domains characterized as liquid-ordered (lo), such as rafts or caveolae, or liquid-disordered (ld). To test for domain–dependent shear sensitivity, we used time-correlated single photon counting instrumentation to assess the photophysics and dynamics of the domain-selective lipid analogues DiI-C12 and DiI-C18 in endothelial cells subjected to physiological fluid shear stress. Under static conditions, DiI-C12 fluorescence lifetime was less than DiI-C18 lifetime and the diffusion coefficient of DiI-C12 was greater than the DiI-C18 diffusion coefficient, confirming that DiI-C12 probes ld, a more fluid membrane environment, and DiI-C18 probes the lo phase. Domains probed by DiI-C12 exhibited an early (10 s) and transient decrease of fluorescence lifetime after the onset of shear while domains probed by DiI-C18 exhibited a delayed decrease of fluorescence lifetime that was sustained for the 2 min the cells were subjected to flow. The diffusion coefficient of DiI-C18 increased after shear imposition, while that of DiI-C12 remained constant. Determination of the number of molecules (N) in the control volume suggested that DiI-C12-labeled domains increased in N immediately after step-shear, while N for DiI-C18-stained membrane transiently decreased. These results demonstrate that membrane microdomains are differentially sensitive to fluid shear stress.

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.