Now showing 1 - 10 of 10
  • Publication
    Neuron Sub-Populations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment
    (American Chemical Society, 2015) ; ;
    Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound proteins that steer elongating axons to their target tissues. Netrin-1, the best characterized member of the Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in colorectal cancer (DCC), a well-established receptor of Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca2+ influx and the basal cytosolic Ca2+ levels regulated the effect of Netrin-1, for example, Ca2+ efflux from the endoplasmic reticulum due to the activation of Ryanodine channels blocked Netrin-1-induced axon slowdown. Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal Netrin-1 response.
      585Scopus© Citations 11
  • Publication
    A microfluidic dual gradient generator for conducting cell-based drug combination assays
    We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays.
      438Scopus© Citations 20
  • Publication
    Bio-Nano-Magnetic Materials for Localized Mechanochemical Stimulation of Cell Growth and Death
    Magnetic nanoparticles are promising new tools for therapeutic applications, such as magnetic nanoparticle hyperthermia therapy and targeted drug delivery. Recent in vitro studies have demonstrated that a force application with magnetic tweezers can also affect cell fate, suggesting a therapeutic potential for magnetically modulated mechanical stimulation. The magnetic properties of nanoparticles that induce physical responses and the subtle responses that result from mechanically induced membrane damage and/or intracellular signaling are evaluated. Magnetic particles with various physical, geometric, and magnetic properties and specific functionalization can now be used to apply mechanical force to specific regions of cells, which permit the modulation of cellular behavior through the use of spatially and time controlled magnetic fields. On one hand, mechanochemical stimulation has been used to direct the outgrowth on neuronal growth cones, indicating a therapeutic potential for neural repair. On the other hand, it has been used to kill cancer cells that preferentially express specific receptors. Advances made in the synthesis and characterization of magnetic nanomaterials and a better understanding of cellular mechanotransduction mechanisms may support the translation of mechanochemical stimulation into the clinic as an emerging therapeutic approach.
      617Scopus© Citations 38
  • Publication
    Advances in magnetic tweezers for single molecule and cell biophysics
    (Royal Society of Chemistry, 2014-11) ;
    Magnetic tweezers (MTW) enable highly accurate forces to be transduced to molecules to study mechanotransduction at the molecular or cellular level. We review recent MTW studies in single molecule and cell biophysics that demonstrate the flexibility of this technique. We also discuss technical advances in the method on several fronts, i.e., from novel approaches for the measurement of torque to multiplexed biophysical assays. Finally, we describe multi-component nanorods with enhanced optical and magnetic properties and discuss their potential as future MTW probes.
      504Scopus© Citations 65
  • Publication
    Flow enhanced non-linear magnetophoretic separation of beads based on magnetic susceptibility
    (Royal Society of Chemistry, 2013-08-20) ; ; ;
    Magnetic separation provides a rapid and efficient means of isolating biomaterials from complex mixtures based on their adsorption on superparamagnetic (SPM) beads. Flow enhanced non-linear magnetophoresis (FNLM) is a high-resolution mode of separation in which hydrodynamic and magnetic fields are controlled with micron resolution to isolate SPM beads with specific physical properties. In this article we demonstrate that a change in the critical frequency of FNLM can be used to identify beads with magnetic susceptibilities between 0.01 and 1.0 with a sensitivity of 0.01 Hz(-1). We derived an analytical expression for the critical frequency that explicitly incorporates the magnetic and non-magnetic composition of a complex to be separated. This expression was then applied to two cases involving the detection and separation of biological targets. This study defines the operating principles of FNLM and highlights the potential for using this technique for multiplexing diagnostic assays and isolating rare cell types.
      355Scopus© Citations 20
  • Publication
    Charge and topography patterned lithium niobate provides physical cues to fluidically isolated cortical axons
    In vitro devices that combine chemotactic and physical cues are needed for understanding how cells integrate different stimuli. We explored the suitability of lithium niobate (LiNbO3), a transparent ferroelectric material that can be patterned with electrical charge domains and micro/nanotopography, as a neural substrate. On flat LiNbO3 z-surfaces with periodically alternating charge domains, cortical axons are partially aligned with domain boundaries. On submicron-deep etched trenches, neurites are aligned with the edges of the topographical features. Finally, we bonded a bicompartmental microfluidic chip to LiNbO3 surfaces patterned by etching, to create isolated axon microenvironments with predefined topographical cues. LiNbO3 is shown to be an emerging neuron culture substrate with tunable electrical and topographical properties that can be integrated with microfluidic devices, suitable for studying axon growth and guidance mechanisms under combined topographical/chemical stimuli.
      394Scopus© Citations 16
  • Publication
    Mechanochemical Stimulation of MCF7 Cells with Rod-Shaped Fe-Au Janus Particles Induces Cell Death through Paradoxical Hyperactivation of ERK
    Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe–Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG–nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG–nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to 'active zones,' dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles.
      621Scopus© Citations 25
  • Publication
    In vitro study of the interaction of heregulin-functionalized magnetic-optical nanorods with MCF7 and MDA-MB- 231 cells
    Multifunctional nanoparticles that actively target specific cells are promising tools for cancer diagnosis and therapy. In this article we review the synthesis and surface chemistry of Fe–Au nanorods and their characterization using microscopy. The diameter of the rods used in this study was selected to be 150–200 nm so that they did not enter the cells. The 80 nm-long Au tips of the nanorods were functionalized with heregulin (HRG), and the micron-long Fe portion was coated with a poly(ethylene glycol) monolayer to minimize non-specific interactions. Nanorods functionalized with HRG were found to preferentially bind to MCF7 cells that express high levels of the receptor tyrosine-protein kinase ErbB2/3. Magnetic tweezers measurements were used to characterize the kinetic properties of the bond between the HRG on the rods and ErbB2/3 on the surface of the cells. The strong magnetization of Fe–Au nanorods makes them excellent candidates for in-vitro and in-vivo imaging, and magnetic therapeutic applications targeting cancer cells in circulation.
      525Scopus© Citations 1
  • Publication
    Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells
    Nonlinear magnetophoresis (NLM) is a powerful approach for on-chip transport and separation of superparamagnetic (SPM) beads, based on a travelling magnetic field wave generated by the combination of a micromagnet array (MMA) and an applied rotating magnetic field. Here, we present two novel MMA designs that allow SPM beads to be focused, sorted, and separated on-chip. Converging MMAs were used to rapidly collect the SPM beads from a large region of the chip and focus them into synchronized lines. We characterise the collection efficiency of the devices and demonstrate that they can facilitate on-chip analysis of populations of SPM beads using a single-point optical detector. The diverging MMAs were used to control the transport of the beads and to separate them based on their size. The separation efficiency of these devices was determined by the orientation of the magnetisation of the micromagnets relative to the external magnetic field and the size of the beads relative to that of micromagnets. By controlling these parameters and the rotation of the external magnetic field we demonstrated the controlled transport of SPM bead-labelled single MDA-MB-231 cells. The use of these novel MMAs promises to allow magnetically-labelled cells to be efficiently isolated and then manipulated on-chip for analysis with high-resolution chemical and physical techniques.
      376Scopus© Citations 9
  • Publication
    Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Netrin-1 modulates axonal growth direction and speed. Its best characterized receptor, Deleted in Colorectal Cancer (DCC), is localized to growth cones, but also observed in the cell bodies. We hypothesized that cell bodies sense Netrin-1 and contribute to axon growth rate modulation, mediated by the second messenger system. We cultured mouse cortical neurons in microfluidic devices to isolate distal axon and cell body microenvironments. Compared to isolated axonal treatment, global Netrin-1 treatment decreased the axon elongation rate and affected the dynamics of total and membranous DCC, calcium, and cyclic nucleotides. Signals induced by locally applied Netrin-1 propagated in both anterograde and retrograde directions, demonstrated by the long-range increase in DCC and by the increased frequency of calcium transients in cell bodies, evoked by axonal Netrin-1. Blocking the calcium efflux from endoplasmic reticulum suppressed the membranous DCC response. Our findings support the notion that neurons sense Netrin-1 along their entire lengths in making axonal growth decisions.
      284Scopus© Citations 11