Tanmay P. Lele

Mechanical Forces Alter Zyxin Unbinding Kinetics Within Focal Adhesions of Living Cells

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant kOFF of individual GFP‐labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques—chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices—increases the kOFF of the focal adhesion protein zyxin. In contrast, the kOFF of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force‐dependent increases in zyxins kOFF that occur over seconds are sufficient to quantitatively predict large‐scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force‐dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells. J. Cell. Physiol. 207: 187–194, 2006.

The control of cell adhesion and viability by zinc oxide nanorods

The ability to control the behavior of cells that interact with implanted biomaterials is desirable for the success of implanted devices such as biosensors or drug delivery devices. There is a need to develop materials that can limit the adhesion and viability of cells on implanted biomaterials. In this study, we investigated the use of zinc oxide (ZnO) nanorods for modulating the adhesion and viability of NIH 3T3 fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells. Cells adhered far less to ZnO nanorods than the corresponding ZnO flat substrate. The few cells that adhered to ZnO nanorods were rounded and not viable compared to the flat ZnO substrate. Cells were unable to assemble focal adhesions and stress fibers on nanorods. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Time-lapse imaging revealed that cells that initially adhered to nanorods were not able to spread. This suggests that it is the lack of initial spreading, rather than long-term exposure to ZnO that causes cell death. We conclude that ZnO nanorods are potentially useful as an adhesion-resistant biomaterial capable of reducing viability in anchorage-dependent cells.

Actomyosin Tension Exerted on the Nucleus through Nesprin-1 Connections Influences Endothelial Cell Adhesion, Migration, and Cyclic Strain-Induced Reorientation

Endothelial cell polarization and directional migration is required for angiogenesis. Polarization and motility requires not only local cytoskeletal remodeling but also the motion of intracellular organelles such as the nucleus. However, the physiological significance of nuclear positioning in the endothelial cell has remained largely unexplored. Here, we show that siRNA knockdown of nesprin-1, a protein present in the linker of nucleus to cytoskeleton complex, abolished the reorientation of endothelial cells in response to cyclic strain. Confocal imaging revealed that the nuclear height is substantially increased in nesprin-1 depleted cells, similar to myosin inhibited cells. Nesprin-1 depletion increased the number of focal adhesions and substrate traction while decreasing the speed of cell migration; however, there was no detectable change in nonmuscle myosin II activity in nesprin-1 deficient cells. Together, these results are consistent with a model in which the nucleus balances a portion of the actomyosin tension in the cell. In the absence of nesprin-1, actomyosin tension is balanced by the substrate, leading to abnormal adhesion, migration, and cyclic strain-induced reorientation.

Contributions of surface topography and cytotoxicity to the macrophage response to zinc oxide nanorods.

Macrophages associated with implanted biomaterials are primary mediators of chronic inflammation and foreign body reaction to the implant. Hence, various approaches have been investigated to modulate macrophage interactions with biomaterial surfaces to mitigate inflammatory responses. Nanostructured materials possess unique surface properties, and nanotopography has been reported to modulate cell adhesion and viability in a cell type-dependent manner. Zinc oxide (ZnO) has been investigated in a number of biomedical applications and surfaces presenting well-controlled nanorod structures of ZnO have recently been developed. In order to investigate the influence of nanotopography on macrophage adhesive response, we evaluated macrophage adhesion and viability on ZnO nanorods, compared to a relatively flat sputtered ZnO controls and using glass substrates for reference. We found that although macrophages are capable of initially adhering to and spreading on ZnO nanorod substrates, the number of adherent macrophages on ZnO nanorods was reduced compared to ZnO flat substrate and glass. Additionally adherent macrophage number on ZnO flat substrate was reduced as compared to glass. While these data suggest nanotopography may modulate macrophage adhesion, reduced cell viability on both sputtered and nanorod ZnO substrate indicates appreciable toxicity associated with ZnO. Cell death was apparently not apoptotic, given the lack of activated caspase-3 immunostaining. A decrease in viable macrophage numbers when ZnO substrates were present in the same media verified the role of ZnO substrate dissolution, and dissolved levels of Zn in culture media were quantified. In order to determine long-term physiological responses, ZnO nanorod-coated and sputtered ZnO-coated polyethylene terephthalate (PET) discs were implanted subcutaneously in mice for 14 d. Upon implantation, both ZnO-coated discs resulted in a discontinuous cellular fibrous capsule indicative of unresolved inflammation, in contrast to uncoated PET discs, which resulted in typical foreign body capsule formation. In conclusion, although ZnO substrates presenting nanorod topography have previously been shown to modulate cellular adhesion in a topography-dependent fashion for specific cell types, this work demonstrates that for primary murine macrophages, cell adhesion and viability correlate to both nanotopography and toxicity of dissolved Zn, parameters which are likely interdependent.

Prostate specific antigen detection using AlGaN∕GaN high electron mobility transistors

Antibody-functionalized Au-gated AlGaN∕GaN high electron mobility transistors (HEMTs) were used to detect prostate specific antigen (PSA). The PSA antibody was anchored to the gate area through the formation of carboxylate succinimdyl ester bonds with immobilized thioglycolic acid. The AlGaN∕GaN HEMT drain-source current showed a rapid response of less than 5s when target PSA in a buffer at clinical concentrations was added to the antibody-immobilized surface. The authors could detect a wide range of concentrations from 10pg∕mlto1μg∕ml. The lowest detectable concentration was two orders of magnitude lower than the cutoff value of PSA measurements for clinical detection of prostate cancer. These results clearly demonstrate the promise of portable electronic biological sensors based on AlGaN∕GaN HEMTs for PSA screening.

A multi-modular tensegrity model of an actin stress fiber

Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on the extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model can also explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors and represent a new handle on multi-scale modeling of living materials.

Investigating complexity of protein-protein interactions in focal adhesions

The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant k(OFF) ranged over an order of magnitude from 0.009+/-0.001/s for talin to 0.102+/-0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.

Fast electrical detection of Hg(II) ions with AlGaN∕GaN high electron mobility transistors

Bare Au gated and thioglycolic acid functionalized Au-gated AlGaN∕GaN high electron mobility transistors (HEMTs) were used to detect mercury (II) ions. Fast detection of less than 5s was achieved for thioglycolic acid functionalized sensors. This is the shortest response time ever reported for mercury detection. Thioglycolic acid functionalized Au-gated AlGaN∕GaN HEMT based sensors showed 2.5 times larger response than bare Au-gated based sensors. The sensors were able to detect mercury (II) ion concentration as low as 10−7M. The sensors showed an excellent sensing selectivity of more than 100 for detecting mercury ions over sodium or magnesium ions. The dimensions of the active area of the sensor and the entire sensor chip are 50×50μm2 and 1×5mm2, respectively. Therefore, portable, fast response, and wireless based heavy metal ion detectors can be realized with AlGaN∕GaN HEMT based sensors.

Randomly oriented, upright SiO2 coated nanorods for reduced adhesion of mammalian cells.

Cell interactions with nanostructures are of broad interest because of applications in controlling tissue response to biomedical implants. Here we show that dense and upright SiO2 coated nanorods nearly eliminate cell adhesion in fibroblasts and endothelial cells. The lack of adhesion is not due to a decrease in matrix protein adsorption on the nanostructures, but rather an inability of cells to assemble focal adhesions. Using spatially patterned nanorods, we show that cells display a preference for flat regions of the surface. Our results support a model in which interfering with nanoscale spacing of ligated integrins results in reduced cell adhesion and subsequent cell death. We propose that dense monolayers of nanorods are a promising nanotechnology for preventing mammalian cell fouling of biomaterials.

Tools to study cell mechanics and mechanotransduction.

Analysis of how cells sense and respond to mechanical stress has been limited by the availability of techniques that can apply controlled mechanical forces to living cells while simultaneously measuring changes in cell and molecular distortion, as well as alterations of intracellular biochemistry. We have confronted this challenge by developing new engineering methods to measure and manipulate the mechanical properties of cells and their internal cytoskeletal and nuclear frameworks, and by combining them with molecular cell biological techniques that rely on microscopic analysis and real-time optical readouts of biochemical signaling. In this chapter, we describe techniques like microcontact printing, magnetic twisting cytometry, and magnetic pulling cytometry that can be systematically used to study the molecular basis of cellular mechanotransduction.