Direct measurement of cortical force generation and polarization in a living parasite.

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality but becomes directed toward the rear of the cell after a period of time. The transition from nondirectional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.

Integration of acoustic radiation force and optical imaging for blood plasma clot stiffness measurement.

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood's transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 µm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Polarized actin structural dynamics in response to cyclic uniaxial stretch.

Endothelial cell (EC) alignment to directional flow or stretch supports anti-inflammatory functions, but mechanisms controlling polarized structural adaptation in response to physical cues remain unclear. This study aimed to determine whether factors associated with early actin edge ruffling implicated in cell polarization are prerequisite for stress fiber (SF) reorientation in response to cyclic uniaxial stretch. Time-lapse analysis of EGFP-actin in confluent ECs showed that onset of either cyclic uniaxial or equibiaxial stretch caused a non-directional increase in edge ruffling. Edge activity was concentrated in a direction perpendicular to the stretch axis after 60 min, consistent with the direction of SF alignment. Rho-kinase inhibition caused reorientation of both stretch-induced edge ruffling and SF alignment parallel to the stretch axis. Arp2/3 inhibition attenuated stretch-induced cell elongation and disrupted polarized edge dynamics and microtubule organizing center reorientation, but it had no effect on the extent of SF reorientation. Disrupting localization of p21-activated kinase (PAK) did not prevent stretch-induced SF reorientation, suggesting that this Rac effector is not critical in regulating stretch-induced cytoskeletal remodeling. Overall, these results suggest that directional edge ruffling is not a primary mechanism that guides SF reorientation in response to stretch; the two events are coincident but not causal.

Cellular and Molecular Bioengineering: A Tipping Point.

In January of 2011, the Biomedical Engineering Society (BMES) and the Society for Physical Regulation in Biology and Medicine (SPRBM) held its inaugural Cellular and Molecular Bioengineering (CMBE) conference. The CMBE conference assembled worldwide leaders in the field of CMBE and held a very successful Round Table discussion among leaders. One of the action items was to collectively construct a white paper regarding the future of CMBE. Thus, the goal of this report is to emphasize the impact of CMBE as an emerging field, identify critical gaps in research that may be answered by the expertise of CMBE, and provide perspectives on enabling CMBE to address challenges in improving human health. Our goal is to provide constructive guidelines in shaping the future of CMBE.

A Semi-Automatic Method for Image Analysis of Edge Dynamics in Living Cells.

Spatial asymmetry of actin edge ruffling contributes to the process of cell polarization and directional migration, but mechanisms by which external cues control actin polymerization near cell edges remain unclear. We designed a quantitative image analysis strategy to measure the spatiotemporal distribution of actin edge ruffling. Time-lapse images of endothelial cells (ECs) expressing mRFP-actin were segmented using an active contour method. In intensity line profiles oriented normal to the cell edge, peak detection identified the angular distribution of polymerized actin within 1 µm of the cell edge, which was localized to lamellipodia and edge ruffles. Edge features associated with filopodia and peripheral stress fibers were removed. Circular statistical analysis enabled detection of cell polarity, indicated by a unimodal distribution of edge ruffles. To demonstrate the approach, we detected a rapid, nondirectional increase in edge ruffling in serum-stimulated ECs and a change in constitutive ruffling orientation in quiescent, nonpolarized ECs. Error analysis using simulated test images demonstrate robustness of the method to variations in image noise levels, edge ruffle arc length, and edge intensity gradient. These quantitative measurements of edge ruffling dynamics enable investigation at the cellular length scale of the underlying molecular mechanisms regulating actin assembly and cell polarization.

A stretching device for high-resolution live-cell imaging.

Several custom-built and commercially available devices are available to investigate cellular responses to substrate strain. However, analysis of structural dynamics by microscopy in living cells during stretch is not readily feasible. We describe a novel stretch device optimized for high-resolution live-cell imaging. The unit assembles onto standard inverted microscopes and applies constant magnitude or cyclic stretch at physiological magnitudes to cultured cells on elastic membranes. Interchangeable modular indenters enable delivery of equibiaxial and uniaxial stretch profiles. Strain analysis performed by tracking fluorescent microspheres adhered onto the substrate demonstrated reproducible application of stretch profiles. In endothelial cells transiently expressing enhanced green fluorescent protein (EGFP)-vimentin and paxillin-DsRed2 and subjected to constant magnitude equibiaxial stretch, the two-dimensional strain tensor demonstrated efficient transmission through the extracellular matrix and focal adhesions. Decreased transmission to the intermediate filament network was measured, and a heterogeneous spatial distribution of maximum stretch magnitude revealed discrete sites of strain focusing. Spatial correlation of vimentin and paxillin displacement vectors provided an estimate of the extent of mechanical coupling between the structures. Interestingly, switching the spatial profile of substrate strain reveals that actin-mediated edge ruffling is not desensitized to repeated mechanostimulation. These initial observations show that the stretch device is compatible with live-cell microscopy and is a novel tool for measuring dynamic structural remodeling under mechanical strain.

Cell Structure Controls Endothelial Cell Migration under Fluid Shear Stress.

Cobblestone-shaped endothelial cells in confluent monolayers undergo triphasic mechanotaxis in response to steady unidirectional shear stress, but cells that are elongated and aligned on micropatterned substrates do not change their migration behavior in response to either perpendicular or parallel flow. Whether mechanotaxis of micropatterned endothelial cell layers is suppressed by elongated cytoskeletal structure or limited availability of adhesion area remains unknown. In this study, cells were examined on wide (100-200 µm) micropatterned lines after onset of shear stress. Cells in center regions of the lines exhibited cobblestone morphology and triphasic mechanotaxis behavior similar to that in unpatterned monolayers, whereas cells along the edges migrated parallel to the line axis regardless of the flow direction. When scratch wounds were created perpendicular to the micropatterned lines, the cells became less elongated before migrating into the denuded area. In sparsely populated lines oriented perpendicular to the flow direction, elongated cells along the upstream edge migrated parallel to the edge for 7 h before migrating parallel to the shear stress direction, even though adhesion area existed in the downstream direction. Thus, cytoskeletal structure and not available adhesion area serves as the dominant factor in determining whether endothelial mechanotaxis occurs in response to shear stress.

Semiconductor nanoparticles as energy mediators for photosensitizer-enhanced radiotherapy.

PURPOSE: It has been proposed that quantum dots (QDs) can be used to excite conjugated photosensitizers and produce cytotoxic singlet oxygen. To study the potential of using such a conjugate synergistically with radiotherapy to enhance cell killing, we investigated the energy transfer from megavoltage (MV) X-rays to a photosensitizer using QDs as the mediator and quantitated the enhancement in cell killing. METHODS AND MATERIALS: The photon emission efficiency of QDs on excitation by 6-MV X-rays was measured using dose rates of 100-600 cGy/min. A QD-Photofrin conjugate was synthesized by formation of an amide bond. The role of Förster resonance energy transfer in the energy transferred to the Photofrin was determined by measuring the degree of quenching at different QD/Photofrin molar ratios. The enhancement of H460 human lung carcinoma cell killing by radiation in the presence of the conjugates was studied using a clonogenic survival assay. RESULTS: The number of visible photons generated from QDs excited by 6-MV X-rays was linearly proportional to the radiation dose rate. The Förster resonance energy transfer efficiency approached 100% as the number of Photofrin molecules conjugated to the QDs increased. The combination of the conjugate with radiation resulted in significantly lower H460 cell survival in clonogenic assays compared with radiation alone. CONCLUSION: The novel QD-Photofrin conjugate shows promise as a mediator for enhanced cell killing through a linear and highly efficient energy transfer from X-rays to Photofrin.

Micropatterned structural control suppresses mechanotaxis of endothelial cells.

Vascular endothelial cell migration is critical in many physiological processes including wound healing and stent endothelialization. To determine how preexisting cell morphology influences cell migration under fluid shear stress, endothelial cells were preset in an elongated morphology on micropatterned substrates, and unidirectional shear stress was applied either parallel or perpendicular to the cell elongation axis. On micropatterned 20-microm lines, cells exhibited an elongated morphology with stress fibers and focal adhesion sites aligned parallel to the lines. On 115-microm lines, cell morphology varied as a function of distance from the line edge. Unidirectional shear stress caused unpatterned cells in a confluent monolayer to exhibit triphasic mechanotaxis behavior. During the first 3 h, cell migration speed increased in a direction antiparallel to the shear stress direction. Migration speed then slowed and direction became spatially heterogeneous. Starting 11-12 h after the onset of shear stress, the unpatterned cells migrated primarily in the downstream direction, and migration speed increased significantly. In contrast, mechanotaxis was suppressed after the onset of shear stress in cells on micropatterned lines during the same time period, for the cases of both parallel and perpendicular flow. The directional persistence time was much longer for cells on the micropatterned lines, and it decreased significantly after flow onset. Migration trajectories were highly correlated among micropatterned cells within a three-cell neighborhood, and shear stress disrupted this spatially correlated migration behavior. Thus, presetting structural morphology may interfere with mechanisms of sensing local physical cues, which are critical for establishing mechanotaxis in response to hemodynamic shear stress.

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells.

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm(2)). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm(2) followed by 15 dyn/cm(2) 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility.

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.

Mapping the dynamics of shear stress-induced structural changes in endothelial cells.

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.

Designing a nano-interface in a microfluidic chip to probe living cells: challenges and perspectives.

Nanotechnology-based materials are beginning to emerge as promising platforms for biomedical analysis, but measurement and control at the cell-chip interface remain challenging. This idea served as the basis for discussion in a focus group at the recent National Academies Keck Futures Initiative. In this Perspective, we first outline recent advances and limitations in measuring nanoscale mechanical, biochemical, and electrical interactions at the interface between biomaterials and living cells. Second, we present emerging experimental and conceptual platforms for probing living cells with nanotechnology-based tools in a microfluidic chip. Finally, we explore future directions and critical needs for engineering the cell-chip interface to create an integrated system capable of high-resolution analysis and control of cellular physiology.

Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells.

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Mechanisms of mechanotransduction.

Essentially all organisms from bacteria to humans are mechanosensitive. Physical forces regulate a large array of physiological processes, and dysregulation of mechanical responses contributes to major human diseases. A survey of both specialized and widely expressed mechanosensitive systems suggests that physical forces provide a general means of altering protein conformation to generate signals. Specialized systems differ mainly in having acquired efficient mechanisms for transferring forces to the mechanotransducers.

Loss of PECAM-1 function impairs alveolarization.

The final stage of lung development in humans and rodents occurs principally after birth and involves the partitioning of the large primary saccules into smaller air spaces by the inward protrusion of septae derived from the walls of the saccules. Several observations in animal models implicate angiogenesis as critical to this process of alveolarization, but all anti-angiogenic treatments examined to date have resulted in endothelial cell (EC) death. We therefore targeted the function of platelet endothelial cell adhesion molecule, (PECAM-1), an EC surface molecule that promotes EC migration and has been implicated in in vivo angiogenesis. Administration of an anti-PECAM-1 antibody that inhibits EC migration, but not proliferation or survival in vitro, disrupted normal alveolar septation in neonatal rat pups without reducing EC content. Three-dimensional reconstruction of lungs showed that pups treated with a blocking PECAM-1 antibody had remodeling of more proximal branches resulting in large tubular airways. Subsequent studies in PECAM-1-null mice confirmed that the absence of PECAM-1 impaired murine alveolarization, without affecting EC content, proliferation, or survival. Further, cell migration was reduced in lung endothelial cells isolated from these mice. These data suggest that the loss of PECAM-1 function compromises postnatal lung development and provide evidence that inhibition of EC function, in contrast to a loss of viable EC, inhibits alveolarization.

Molecular control of cytoskeletal mechanics by hemodynamic forces.

The endothelium at the interface between blood and tissue acts as a primary transducer of local hemodynamic forces into signals that maintain physiological function or initiate pathological processes in vessel walls. Rapid intracellular spatial gradients of structural dynamics and signaling molecule activity suggest that mechanical cues at the molecular level guide cellular mechanotransduction and adaptation to shear stress profiles.