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Cells are sensitive to mechanical cues from the ECM such as, stiffness, topography, and roughness. Tissue stiffening due to ageing or diseases has been shown to affect cell proliferation, differentiation, migration, and apoptosis, but the mechanosensors that respond to ECM stiffness changes have not yet been fully understood. Recent studies on mechanosensitive Piezo1 channels have shown that these channels are sensitive to ECM cues, such as mechanical confinements implemented by micropatterns. We hypothesize that Piezo1 could serve as a force sensor for local ECM stiffness. Using a ‘2-well PDMS chip’ consisting of substrates of different stiffness, namely, hard (∼1000 kPa) and soft (∼0.1 kPa), we studied the role of Piezo1 on stiffness-dependent morphology changes in epithelial cells. The results show that cells respond to substrate stiffness variations with profound cytoskeletal reorganization and moderate changes in their spreading area and shape. Cells on the hard substrates were only ∼20% larger than that on soft substrates within the same culture period of 2.5 hrs and at a similar confluency. However, cells on hard substrates show abundant F-actin bundles that are reorganized to peripheral actin rings on soft substrates. Inhibition of Piezo1 with GsMTx4 or Gd3+, largely reduced the formation of thick actin bundles on hard substrates. Activation of Piezo1 with specific agonist, Yoda1, enhanced the formation of actin bundles. These results indicate that the epithelial cells’ response to ECM stiffness is mediated by Piezo1 through its function of permeating Ca2+ ions. This study demonstrates that Piezo1 could be an ECM mechanosensor in epithelial cells.

Adherent cells utilize local environmental cues to make decisions on their growth and movement. We have previously shown that HEK293 cells grown on the fibronectin stripe patterns were elongated. Here we show that Piezo1 function is involved in cell spreading. Piezo1 expressing HEK cells plated on fibronectin stripes elongated, while a knockout of Piezo1 eliminated elongation. Inhibiting Piezo1 conductance using GsMTx4 or Gd3+ blocked cell spreading, but the cells grew thin tail-like extensions along the patterns. Images of GFP-tagged Piezo1 showed plaques of Piezo1 moving to the extrusion edges, co-localized with focal adhesions. Surprisingly, in non-spreading cells Piezo1 was located primarily on the nuclear envelope. Inhibiting the Rho-ROCK pathway also reversibly inhibited cell extension indicating that myosin contractility is involved. The growth of thin extrusion tails did not occur in Piezo1 knockout cells suggesting that Piezo1 may have functions besides acting as a cation channel.

Traumatic brain injury (TBI) is a major risk factor for development of neurodegenerative disorders later in life. Short, repetitive, mechanical impacts can lead to pathology that appears days or months later. The cells have a physical "memory" of mechanical events. The origin of this memory is not known. To examine the properties of this memory, we used a microfluidic chip to apply programmed fluid shear pulses to adherent adult rat astrocytes. These caused a transient rise in intracellular Ca

Traumatic brain injury (TBI) elevates Abeta (Aβ) peptides in the brain and cerebral spinal fluid. Aβ peptides are amphipathic molecules that can modulate membrane mechanics. Because the mechanosensitive cation channel PIEZO1 is gated by membrane tension and curvature, it prompted us to test the effects of Aβ on PIEZO1. Using precision fluid shear stress as a stimulus, we found that Aβ monomers inhibit PIEZO1 at femtomolar to picomolar concentrations. The Aβ oligomers proved much less potent. The effect of Aβs on Piezo gating did not involve peptide-protein interactions since the D and L enantiomers had similar effects. Incubating a fluorescent derivative of Aβ and a fluorescently tagged PIEZO1, we showed that Aβ can colocalize with PIEZO1, suggesting that they both had an affinity for particular regions of the bilayer. To better understand the PIEZO1 inhibitory effects of Aβ, we examined their effect on wound healing. We observed that over-expression of PIEZO1 in HEK293 cells increased cell migration velocity ~10-fold, and both enantiomeric Aβ peptides and GsMTx4 independently inhibited migration, demonstrating involvement of PIEZO1 in cell motility. As part of the motility study we examined the correlation of PIEZO1 function with tension in the cytoskeleton using a genetically encoded fluorescent stress probe. Aβ peptides increased resting stress in F-actin, and is correlated with Aβ block of PIEZO1-mediated Ca2+ influx. Aβ inhibition of PIEZO1 in the absence of stereospecific peptide-protein interactions shows that Aβ peptides modulate both cell membrane and cytoskeletal mechanics to control PIEZO1-triggered Ca2+ influx.

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells' lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration.

Adherens junctions (AJs) are a key structural component for tissue organization and function. Under fluid shear stress, AJs exhibit dynamic assembly/disassembly, but how shear stress couples to AJs is unclear. In MDCK cells we measured simultaneously the forces in cytoskeletal α-actinin and the density and length of AJs using a genetically coded optical force sensor, actinin-sstFRET, and fluorescently labeled E-cadherin (E-cad). We found that shear stress of 0.74 dyn/cm2 for 3 h significantly enhanced E-cad expression at cell-cell contacts and this phenomenon has two phases. The initial formation of segregated AJ plaques coincided with a decrease in cytoskeletal tension, but an increase in tension was necessary for expansion of the plaques and the formation of continuous AJs in the later phase. The changes in cytoskeletal tension and reorganization appear to be an upstream process in response to flow since it occurred in both wild type and dominant negative E-cad cells. Disruption of F-actin with a Rho-ROCK inhibitor eliminated AJ growth under flow. These results delineate the shear stress transduction paths in cultured cells, which helps to understand pathology of a range of diseases that involve dysfunction of E-cadherin.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

External mechanical forces can reach the cell nucleus causing changes in nuclear morphology, size and motility. A common explanation is that these forces are transmitted by surrounding cytoskeleton network through its linkage to nuclear envelope; shear stress causes reorganization of cytoskeleton, thus, the changes in nuclear shape. In this study, we measured nuclear shape and intracellular Ca2+ under fluid shear stress in MDCK cells using a parallel plate microfluidic chip. We show that fluid shear stress (1.1 dyn/cm2, 3 hrs) causes significant changes in nuclear shape in cells, from a flat disk shape having larger area to a thicker disk having smaller area. An increase in intracellular Ca2+ is required for shear induced nucleus deformation. Inhibiting Ca2+ influx with GsMTx4 and Gd3+ eliminated Ca2+ influx and abolished the nuclear deformation. The cytoskeleton reorganization occurred in parallel with Ca2+ rise in the cells. Increasing intracellular Ca2+ with thapsigargin that depletes the Ca2+ stores resumed the nuclear deformation. This suggests that shear induced nuclear deformation is a Ca2+ dependent process.

Traumatic brain injury (TBI) refers to brain damage resulting from external mechanical force, such as a blast or crash. Our current understanding of TBI is derived mainly from in vivo studies that show measurable biological effects on neurons sampled after TBI. Little is known about the early responses of brain cells during stimuli and which features of the stimulus are most critical to cell injury. We generated defined shear stress in a microfluidic chamber using a fast pressure servo and examined the intracellular Ca(2+) levels in cultured adult astrocytes. Shear stress increased intracellular Ca(2+) depending on the magnitude, duration, and rise time of the stimulus. Square pulses with a fast rise time (∼2 ms) caused transient increases in intracellular Ca(2+), but when the rise time was extended to 20 ms, the response was much less. The threshold for a response is a matrix of multiple parameters. Cells can integrate the effect of shear force from repeated challenges: A pulse train of 10 narrow pulses (11.5 dyn/cm(2) and 10 ms wide) resulted in a 4-fold increase in Ca(2+) relative to a single pulse of the same amplitude 100 ms wide. The Ca(2+) increase was eliminated in Ca(2+)-free media, but was observed after depleting the intracellular Ca(2+) stores with thapsigargin suggesting the need for a Ca(2+) influx. The Ca(2+) influx was inhibited by extracellular Gd(3+), a nonspecific inhibitor of mechanosensitive ion channels, but it was not affected by the more specific inhibitor, GsMTx4. The voltage-gated channel blockers, nifedipine, diltiazem, and verapamil, were also ineffective. The data show that the mechanically induced Ca(2+) influx commonly associated with neuron models for TBI is also present in astrocytes, and there is a viscoelastic/plastic coupling of shear stress to the Ca(2+) influx. The site of Ca(2+) influx has yet to be determined.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

Microfluidics is an interdisciplinary field intersecting many areas in engineering. Utilizing a combination of physics, chemistry, biology, and biotechnology, along with practical applications for designing devices that use low volumes of fluids to achieve high-throughput screening, is a major goal in microfluidics. Microfluidic approaches allow the study of cells growth and differentiation using a variety of conditions including control of fluid flow that generates shear stress. Recently, Piezo1 channels were shown to respond to fluid shear stress and are crucial for vascular development. This channel is ideal for studying fluid shear stress applied to cells using microfluidic devices. We have developed an approach that allows us to analyze the role of Piezo channels on any given cell and serves as a high-throughput screen for drug discovery. We show that this approach can provide detailed information about the inhibitors of Piezo channels.

Network activity in the brain is associated with a transient increase in extracellular K(+) concentration. The excess K(+) is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1), and/or Na(+)/K(+)-ATPase activity. Their individual contribution to [K(+)]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na(+)/K(+)-ATPase and to resolve their involvement in clearance of extracellular K(+) transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K(+)]o increases above basal levels. Increased [K(+)]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K(+) clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K(+) removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K(+)]o increase. In contrast, inhibition of the different isoforms of Na(+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2β2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K(+) recovery in native hippocampal tissue while Kir4.1 and Na(+)/K(+)-ATPase serve temporally distinct roles.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser(111) and of phosphorylation-dependent gating of AQP4.

During traumatic brain injury, brain cells experience transient shear stresses that alter the forces in structural proteins. An increase in intracellular Ca2+ is seen universally in cells subjected to shear forces, but how are mechanical forces coupled to the Ca2+ influx is not well understood. We used a microfluidic chamber driven with a high-speed pressure servo to generate controlled fluid shear stress to cultured astrocytes, and simultaneously measured the intracellular responses using FRET-based force sensors actinin-cpstFRET and Ca2+ probes jRCaMP1h. We found that fluid shear generated non-uniform stresses in actinin. The time dependent force distribution is highly sensitive to the feature of the stimulus such as amplitude, duration rise time, and the frequencies of stimulation. In cells with weaker stress fibers, a rapid shear pulse (23 dyn/cm2, 2 ms rise time, 400 ms duration) produced an immediate and long-lasting increase in actinin stress at the upstream end of the cell and minimal changes at the downstream end. In contrast, a slow ramp to the same amplitude caused a minimal, and a more uniform increase in actinin stresses. Simultaneous Ca2+ imaging showed that the initial Ca2+ rise began at the upstream end of the cell where there was high strain, and it propagated to the entire cell within ∼4 s. Moreover, the Ca2+ peaked much faster (∼4 s) at the front end and slower in the center of cell body (∼10 s). This behavior occurred in ∼40% cells. In cells with abundant strong actin bundles, there was minimal response to shear stress. The actinin stress increased transiently and uniformly over the entire cell during the period of stimulation, and the Ca2+ rise appeared starting from the cell body. These results suggest that the force distribution directly alter the initial Ca2+ dynamics in different cellular domains. This work was funded by NINDS.

Pathogenic bacteria are one of the major concerns in food industries and water treatment facilities because of their rapid growth and deleterious effects on human health. The development of fast and accurate detection and identification systems for bacterial strains has long been an important issue to researchers. Although confirmative for the identification of bacteria, conventional methods require time-consuming process involving either the test of characteristic metabolites or cellular reproductive cycles. In this paper, we review recent sensing strategies based on micro- and nano-fabrication technology. These technologies allow for a great improvement of detection limit, therefore, reduce the time required for sample preparation. The paper will be focused on newly developed nano- and micro-scaled biosensors, novel sensing modalities utilizing microfluidic lab-on-a-chip, and array technology for the detection of pathogenic bacteria.

We report a microfluidic chip that is capable of measuring volume changes in single cells in real-time. Single eukaryotic cells were immobilized in the sensing area and changes in volume in response to hypotonic challenges and drugs were measured using the electrical impedance method. Experiments on MDCK cells showed that the maximum swelling and the time course of swelling vary between individual cells following hypotonic stimulation. The microfluidic chip allows, rapid and convenient change of solutions, enabling detailed studies of various drugs and chemicals that may play important role in cell physiology at the single cell level.

Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3- or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10x faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of Hg (2+) and Ni (2+) on the water transport via AQPs. Hg (2+) inhibited the water flux in AQP3-expressing cells irreversibly, while Ni (2+) blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs.

Using a microfluidic volume sensor, we studied the dynamic effects of Hg2+ on hypotonic stress-induced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 muM HgCl2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na+ with impermeant NMDG+ in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg2+-induced excess swelling. The volume decrease in the presence of Hg2+ was inhibited by tetraethylammonium and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, blockers of K+ and Cl(-) channels, respectively, suggesting that part of the Hg2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steady-state volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.

We developed a microfluidic chip that provides rapid temperature changes and accurate temperature control of the perfusing solution to facilitate patch-clamp studies. The device consists of a fluid channel connected to an accessible reservoir for cell culture and patch-clamp measurements. A thin-film platinum heater was placed in the flow channel to generate rapid temperature change, and the temperature was monitored using a thin-film resistor. We constructed the thermal chip using SU-8 on a glass wafer to minimize the heat loss. The chip is capable of increasing the solution temperature from bath temperature (20 degrees C) to 80 degrees C at an optimum heating rate of 0.5 degrees C/ms. To demonstrate the ability of the thermal chip, we have conducted on-chip patch-clamp recordings of temperature-sensitive ion channels (TRPV1) transfected HEK293 cells. The heat-stimulated currents were observed using whole-cell and cell-attached patch configurations. The results demonstrated that the chip can provide rapid temperature jumps at the resolution of single-ion channels.

In this paper, we have developed a simple and rapid method for the detection and identification of bacteria using a microfluidic lab-chip. The microfluidic chip utilizes impedance-based measurement to (1) detect cells and (2) identify them when used in conjunction with immobilized monoclonal antibodies. Bacteria in suspension passing through the microfluidic chamber are recognized by antibodies and selectively immobilized on the functionalized glass surface, thereby increasing the measured impedance within the chamber. Continuous perfusion of bacteria suspension through the derivatized chamber not only identifies specific bacteria but also enhances the chamber's detection sensitivity by accumulating bacteria on the chamber wall over time; this approach would be useful for detecting low concentrations of bacteria. To demonstrate this approach, we showed that the prototype sensor could detect 9 × 105 CFU mL−1 E. coli (BL21(DE3)) in the solution by consecutive perfusions. The chip sensitivity with immobilized bacteria is governed by height of sensing chamber, and ∼104 CFU mL−1 of E. coli could easily be detected when a shallower chamber (2 μm high) was used. The selectivity of the sensor was tested using a suspension of two bacterial strains, E. coli and M. catarrhalis. The sensor chip is simple to use, requires minuscule samples, and eliminates extensive cell culture processes. Development of more advanced lab-chips with multiple chambers containing different antibodies that allow simultaneous detection of different bacteria strains will be a natural extension of this work.

Accurate measurement and control of flow properties are essential for microfluidic lab-chips. In this paper, we demonstrate an electrolytic-bubble-based approach to directly measure flow rate in microfluidic arteries. By simultaneously generating two gas bubbles electrochemically along a channel, we can measure the pressure difference (and thus flow rate) in real time. A prototype chip that measures flow rate was fabricated on silicon. The sensor was characterized for a functional microfluidic system with inlet pressure ranging from 108 to 135 kPa. The flow-meter performance was compared with computational fluid dynamics simulations and was also calibrated against other direct experiments. The impedance-based flow-rate measurements are easily achieved on a chip with a simple electronic circuit. The described approach can be integrated into any fluid circuit, particularly microfluidic channels that are too small to use off-shelf flow meters.

Adherens junctions (AJs) play an important role in cell-cell mechanical coupling and in maintaining tissue organization. Under chronic flow, epithelial cells adapt by strengthening cell-cell junctions in order to maintain the integrity of epithelia. How forces due to fluid flow are transmitted to E-cadherin and cause the dynamic remodeling of AJs is unclear. In this study we simultaneously measured AJ dynamics and cytoskeletal stresses and reorganization in MDCK cells under flow. The cells were co-expressed with fluorescently labelled E-cadherin and stress sensitive α-actinin FRET sensor for measurements. A fluid shear stress of 0.7dyn/cm2 for 3 hours caused an increase in expression of E-cadherin, formation of new AJs and strengthening of pre-existing AJs leading to a continuous distribution of cell-cell junctions. This increase in cell-cell junctions is correlated with the formation of strong peripheral actin ring due to cytoskeleton reorganization. The average cytoskeletal stress at adherens junctions shows multi-phase changes under flow, which closely links to the dynamic changes of AJs. These results provide the direct evidence that the shear stress induced cytoskeletal forces play an important role on cell-cell adhesions.

Cells respond to fluid shear stress through dynamic processes involving changes in actomyosin and other cytoskeletal stresses, remodeling of cell adhesions, and cytoskeleton reorganization. In this study we simultaneously measured focal adhesion dynamics and cytoskeletal stress and reorganization in MDCK cells under fluid shear stress. The measurements used co-expression of fluorescently labeled paxillin and force sensitive FRET probes of α-actinin. A shear stress of 0.74 dyn/cm2 for 3 hours caused redistribution of cytoskeletal tension and significant focal adhesion remodeling. The fate of focal adhesions is determined by the stress state and stability of the linked actin stress fibers. In the interior of the cell, the mature focal adhesions disassembled within 35–40 min under flow and stress fibers disintegrated. Near the cell periphery, the focal adhesions anchoring the stress fibers perpendicular to the cell periphery disassembled, while focal adhesions associated with peripheral fibers sustained. The diminishing focal adhesions are coupled with local cytoskeletal stress release and actin stress fiber disassembly whereas sustaining peripheral focal adhesions are coupled with an increase in stress and enhancement of actin bundles. The results show that flow induced formation of peripheral actin bundles provides a favorable environment for focal adhesion remodeling along the cell periphery. Under such condition, new FAs were observed along the cell edge under flow. Our results suggest that the remodeling of FAs in epithelial cells under flow is orchestrated by actin cytoskeletal stress redistribution and structural reorganization.

Recent work on magnetic quantum point contacts (QPCs) was discussed. Complete magnetoresistance loops across Co QPCs as small as a single atom was measured. The remarkable feature of these QPCs is the rapid oscillatory decay in magnetoresistance with the increase of contact size. In addition, stepwise or quantum magnetoresistance loops are observed, resulting from varying transmission probability of the available discrete conductance channels because the sample is cycled between the ferromagnetic (F) and antiferromagnetic (AF) aligned states. Quantized conductance combined with spin dependent transmission of electron waves gives rise to a multi-channel system with a quantum domain wall acting as a valve, i.e., a quantum spin-valve. Behavior of a few-atom QPC is built on the behavior of a single-atom QPC and hence the summarization of results as ‘single-atom spintronics’. An evolutionary trace of spin-dependent electron transmission from a single atom to bulk is provided, the requisite hallmarks of artefact-free magnetoresistance is established across a QPC – stepwise or quantum magnetoresistance loops and size dependent oscillatory magnetoresistance.

Magnetic materials reduced to a single atom show unexpected magnetic properties that interact with the spin states of the transmitting electrons and modify the nature of the quantized conductance. This integration of quantized conductance and spin-dependent transport across a magnetic atom gives rise to a multichannel system across which the transmission of electron waves can be regulated by a domain wall acting as a ‘valve’ —a quantum spin-valve. Here we measure complete magnetoresistance loops across magnetic quantum point contacts as small as a single atom, using cobalt. ‘Discrete’ or quantum magnetoresistance loops are observed owing to the varying transmission probability from the available discrete conductance channels. A remarkable feature of these quantum contacts is the discovery of a rapid oscillatory decay in magnetoresistance with increasing contact size. The results provide an evolutionary trace of spin-dependent transport from a single atom to larger ensembles.

This article presents our recent results on magnetic shape memory alloys (SMAs) and reviews some of our relevant past work. By treating the martensite structure as an assembly of microscopic crystalline tiles (twins), the observed micromagnetic structure or 'magnetic mosaic' in these alloys can be explained in terms of a spatially varying axis of the magnetocrystalline anisotropy and magnetostatic interactions across the tiles. A systematic investigation of ferroelastic structural domains of increasing structural complexity leads to a novel concept of 'polymagnets'. Similar to the idea of hierarchical ferroelastic polydomains, polymagnets are hierarchical micromagnetic structures, and parallels the complexity of the underlying ferroelastic polydomains. Preliminary results on the dynamics of the transformation pathways reveal a strong correlation between the underlying microstructure and achievable strain. In addition, reversible shape memory effect has been observed in sputter- deposited Ni-Mn-Ga thin films, and Co-Ni films with controlled composition have been successfully made by electrodeposition.

In this paper we have electrically measured the response of electrolytic gas bubbles to varying pressures in a microfluidic channel. Impedance measurements were used to monitor either the volume or volume change of an electrolytic bubble. Experimental results show that both the bubble size and bubble dissolution rate are sensitive to the applied pressure, and can be readily measured in the fluidic channel. The bubble response to pressures ranging from 101.3 (1 atm) to 112.7 kPa was characterized. Various surface wettabilities were tested, and results show that in a microfluidic channel surface-free energies at the solid–liquid–gas interfaces play an important role in the measured response. Results provide an electrical measurement approach towards pressure or flow rate sensing in microfluidic systems using electrolytic gas bubbles.

Regulation of cell volume was one of the earliest evolutionary demands for life and remains a universal measure of cell metabolism. Since conventional methods to measure cell volume, such as microscopy, are complex and time-consuming, cell volume has not been used as the basis for cell-based screening. We have developed a microfabricated chip that can measure the volume of small numbers of cells in real time with unprecedented resolution. The method is applicable to adherent or suspended populations of cells and membrane-bound organelles. Our prototype device can detect volume changes in a monolayer of tissue-cultured astrocytes responding to anisotonic stimuli of1mOsm. We determined the sensitivity to antibiotics of different E. coli strains in10 min at 24 degrees C. This time can be reduced at higher temperatures enabling on-site clinical testing of infectious agents. Using the chip to screen natural products, we found a peptide in spider venom that inhibits eukaryotic volume regulation at approximately 100pM. The prototype chip made in silicon is inexpensive, reusable, and runs on low-voltage electrical power. The technology can be readily transferred to large arrays in plastic.

Fluid shear stress induced membrane transport such as Ca2+ influx and the activation of ion channels has been widely reported. The shear stress can be mediated by a direct change in bilipid membrane tension and/or by a change in cytoskeletal stress via binding proteins that link channels to actin, but how the shear stress is coupled to ion channels is unclear. Using narrow shear pulse stimuli generated by a pressure servo in a microfluidic chamber, we measured the changes of membrane tension and cytoskeletal protein stress simultaneously in astrocytes. The membrane tension was reported using molecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ) and cytoskeletal tension reported by genetically encoded force probe actinin-cpst-FRET. Our results show that the changes of membrane tension are highly localized and the gradient is relevant to the flow directions. A shear stress pulse (23 dyn/cm2, 400 ms duration) caused a rapid increase in membrane tension at the front edge of the cell with respect to the flow and a decrease in tension (compression) at the distal edge. The rise time was less than 30 ms, and the tension dropped to the initial state within ∼30 ms post stimulus, showing a typical elastic behavior. In contrast, the same shear pulse generated profound and long-lasting tension in cytoskeletal cross-linking proteins α-actinin at the front edge of the cell, and the tension persisted for the entire experimental duration of 60 s. In situ Ca2+ imaging showed that the initial Ca2+ influx was strongly correlated with the region having high cytoskeletal tension, but weakly linked to the bilayer tension. The results suggest the cytoskeletal tension plays primary role in shear stress activated Ca2+ influx. This work was funded by NINDS.

PIEZO1 is a eukaryotic mechanosensitive ion channel that is cation selective and rapidly inactivates. Using fluid shear stress as a stimulus generated in a microfluidic chamber, we have monitored PIEZO activity in the presence of various peptides. To do so, we first developed a cell line that overexpresses the PIEZO1 channel in HEK293T cells. We estimate that there are about 5000 channels per cell. These cells are seeded in the microfluidic chamber and loaded with calcium indicator. A pressure servo is fitted to the chamber and allows control of the applied shear stress stimulus. The calcium change is monitored as an increase in fluorescence. Applying a 10 ms pulse at 13 dyn/cm2, we observed a robust response for the PIEZO1 cell line well above the background observed for HEK293T cells only. Peak response is achieved within a few seconds and is followed by adaptation. Disrupters of the cytoskeleton applied to the cell line, such as cytochalasinD and cholchicine, rendered the PIEZO1 channel unresponsive to the stimulus. The peptide GsMTx4 inhibits PIEZO1 currents. We measured the effect of GsMTx4 on PIEZO1 activity as a way to compare the microfluidic approach to previous work. We applied various concentrations and measure the peak response. The inhibition curve showed a Ki of around 250 nM similar to the value measured previously by patch clamp. Next, we examined the effect of Abeta peptides on PIEZO1 activity. A known attribute of these peptides is their ability to bind membranes. Given PIEZO1 is modulated by membrane tension we wanted to determine how the channel behaved in the presence of Abeta peptides. We prepared the monomer form of the peptide and measured the peak response as a function of peptide concentration. At the same time we prepared the D form of these peptides and measured them as well. Our data show that both the L and D form of Abeta 1-40 and 1-42 inhibit channel function in the fM to pM range. The enantiomeric forms of each peptide were similar in their ability to inhibit PIEZO1 activity. The lack of stereospecific interaction indicates that these peptides modulate activity of the channel by changing the properties of the membrane.This work was funded by NIHLBI and NINDS.

Adherent cells interact with extracellular matrix via cell-substrate contacts at focal adhesions. The dynamic assembly and disassembly of focal adhesions enables cell attachment, migration and growth. While the influence of mechanical forces on the formation and growth of focal adhesions has been widely observed, the force loading on specific proteins at focal adhesion complex is not clear. By co-expressing force sensitive α-actinin FRET probes and fluorescence labeled paxillin in MDCK cells, we have simultaneously observed the time-dependent changes in tension in α-actinin and the dynamics of focal adhesion during cell migration. We show that increase in tension in α-actinin at the focal adhesion coincides with elongation of the adhesion in its growth phase. The enlargement of focal adhesion is through a force sensitive recruitment of α-actinin and paxillin to the adhesion sites. Changes in α-actinin tension and correlated relocation of α-actinin in an active adhesion also guide the growth direction of the adhesion. The results support the model that cytoskeletal tension is coupled to focal adhesion via the linking protein, α-actinin at the adhesion complex. Lysophosphatidic acid caused an immediate increase in α-actinin tension followed by drastic focal adhesion formation and elongation. Application of Rho-ROCK inhibitor, Y27632, resulted in reversible reduction in tension in α-actinin and disassociation of focal adhesion, suggesting the involvement of myosin-II mediated contractile force in the focal adhesion dynamics. These findings suggest that α-actinin not only serves as a physical linker between cytoskeleton and integrin, but also participates in force transmission at adhesion sites to facilitate adhesion׳s growth.

Interaction of cells with extracellular matrix (ECM) regulates cell shape, differentiation and polarity. This effect has been widely observed in cells grown on substrates with various patterned features, stiffness and surface chemistry. It has been postulated that mechanical confinement of cells by the substrate causes a redistribution of tension in cytoskeletal proteins resulting in cytoskeletal reorganization through force sensitive pathways. However, the mechanisms for force transduction during reorganization remain unclear. In this study, using FRET based force sensors we have measured tension in an actin cross-linking protein, α-actinin, and followed reorganization of actin cytoskeleton in real time in HEK cells grown on patterned substrates. We show that the patterned substrates cause a redistribution of tension in α-actinin that coincides with cytoskeleton reorganization. Higher tension was observed in portions of cells where they form bridges across inhibited regions of the patterned substrates; the attachment to the substrate is found to release tension. Real time measurements of α-actinin tension and F-actin arrangement show that an increase in tension coincides with formation of F-actin bundles at the cell periphery during cell-spreading across inhibited regions, suggesting that mechanical forces stimulate cytoskeleton enhancement. Rho-ROCK inhibitor (Y27632) causes reduction in actinin tension followed by retraction of bridged regions. Our results demonstrate that changes in cell shape and expansion over patterned surfaces is a force sensitive process that requires actomyosin contractile force involving Rho-ROCK pathway.

The U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a national user facility, serves the diverse scientific community by providing integrated high-throughput sequencing and computational analysis to enable system-based scientific approaches in support of DOE missions related to clean energy generation and environmental characterization. The JGI Genome Portal (http://genome.jgi.doe.gov) provides unified access to all JGI genomic databases and analytical tools. The JGI maintains extensive data management systems and specialized analytical capabilities to manage and interpret complex genomic data. A user can search, download and explore multiple data sets available for all DOE JGI sequencing projects including their status, assemblies and annotations of sequenced genomes. Here we describe major updates of the Genome Portal in the past 2 years with a specific emphasis on efficient handling of the rapidly growing amount of diverse genomic data accumulated in JGI.

The effect of surface modifying species, Pb, on the nanostructure and resulting giant magnetoresistive properties of symmetric spin valves of the type NiO/Co/Cu/Co/Cu/Co/NiO is investigated, using high-resolution and conventional transmission electron microscopy. When a monolayer of Pb is deposited on the first Co/Cu bilayer, the net ferromagnetic coupling between the middle Co layer and the NiO-pinned Co layers is reduced by an order of magnitude. Nanostructure investigations revealed that, in the absence of Pb, the Co and Cu layers grow coherently over each other within columnar grains. The coherent growth mode of the metal layers in the Pb-free samples gives rise to topographically correlated ferromagnetic films, a requisite if ferromagnetic coupling arises from N\'eel's 'orange-peel' effect. In the presence of Pb, the deposition proceeds by the nucleation of fine, (apparently) randomly oriented nanocrystallites in successive metal layers, instead of a coherent growth mode, thereby disrupting topographic correlation between the adjacent ferromagnetic films, and reducing the net ferromagnetic coupling due to N\'eel"s 'orange-peel' effect. Due to well defined orientation relationships between the adjacent grains in the Pb-free samples, a faceted surface morphology develops, on which the top NiO film is deposited. This is in contrast to the Pb-containing samples which have an irregular, undulatorylike surface. Although not directly observable, the characteristics of Co-Cu interfaces can be inferred from the nature of the top Co surface.

High resolution transmission electron microscopy (HRTEM) was used to investigate the nanostructure of symmetric spin-valves (SSV) of the type NiO/Co/Cu/Co/Cu/Co/NiO. The Co/Cu layers grow coherently over each other within columnar grains, the topography of the columns being conformal with that of the underlying NiO layer. Clear evidence is presented which shows that the middle Co layer switches between antiferromagnetic (AF) ↔ ferromagnetic (F) aligned states by domain wall nucleation and growth; the Co layers, magnetically pinned by NiO, undergo reversal at high fields (48 mT), but no accompanying wall motion was observed. The wall nucleation of the middle Co layer is explained in terms of the presence of local AF-coupled regions arising from fluctuations in Cu layer thickness.

In this paper, we present a microfluidic chip that is capable of measuring electrical conductance through gap junction channels in a 2-dimensional cell sheet. The chip utilizes a tri-stream laminar flow to create a non-conductive sucrose gap between the two conducting solutions so that electrical current can pass across the sucrose gap only through the cells. Using the chip, we tested the effect of a gap junction inhibitor, 2-APB, on the electrical coupling of connexin 43 (Cx43) gap junction channels in NRK-49F cells. We found that 2-APB reversibly blocks the conductivity in a dose-dependent manner. The tri-stream chip further allows us to simultaneously follow the conductance changes and dye diffusion in real time. We show that 2-APB affects both conductance and diffusion, supporting the interpretation that both sets of data reflect the same gap junction activity. The chip provides a generic platform to investigate gap junction properties and to screen drugs that may inhibit or potentiate gap junction transmission.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser(111) and of phosphorylation-dependent gating of AQP4.

Using a microfluidic cell volume sensor we measured the change in the cell volume of Madin-Darby Canine Kidney (MDCK) cells induced by shear stress. An increase in shear stress from 0.2 to 2.0 dyn/cm(2) resulted in a volume decrease to a steady state volume ∼ 20 - 30 % smaller than the initial resting cell volume. Independent experiments based on fluorescence quenching confirmed the volume reduction. This shear-induced cell shrinkage was irreversible on the time scale of the experiment (∼ 30 min). Treatment of 0.1 µM Hg(2+) significantly inhibited the volume decrease, suggesting that the shear-induced cell shrinkage is associated with water efflux through aquaporins. The volume decrease cannot be inhibited by 75 mM TEA, 100 µM DIDS, or 100 µM Gd(3+) suggesting that volume reduction is not directly mediated by K(+) and Cl(-)channels that typically function during regulatory volume decrease (RVD), nor is it through cationic stretch-activated ion channels (SACs). The process also appears to be Ca(2+) independent because it was insensitive to intracellular Ca(2+) level. Since cell volume is determined by the intracellular water content, we postulate that the shear induced reductions in cell volume may arise from increased intracellular hydrostatic pressure as the cell is deformed under flow, which promotes the efflux of water. The increase in internal pressure in a deformable object under the flow is supported by the finite element mechanical model.

Using stress sensitive FRET sensors we have measured cytoskeletal stresses in α-actinin and the associated reorganization of the actin cytoskeleton in cells subjected to chronic shear stress. We show that long-term shear stress reduces the average actinin stress and this effect is reversible with removal of flow. The flow-induced changes in cytoskeletal stresses are found to be dynamic, involving a transient decrease in stress (phase-I), a short-term increase (3-6 min) (Phase-II), followed by a longer-term decrease that reaches a minimum in ~20 min (Phase-III), before saturating. These changes are accompanied by reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles. Blocking mechanosensitive ion channels (MSCs) with Gd(3+) and GsMTx4 (a specific inhibitor) eliminated the changes in cytoskeletal stress and the corresponding actin reorganization, indicating that Ca(2+) permeable MSCs participate in the signaling cascades. This study shows that shear stress induced cell adaptation is mediated via MSCs.