Silver nanoparticles induce neurotoxicity in a human embryonic stem cell-derived neuron and astrocyte network

<p>Silver nanoparticles (AgNPs) are among the most extensively used nanoparticles and are found in a variety of products. This ubiquity leads to inevitable exposure to these particles in everyday life. However, the effects of AgNPs on neuron and astrocyte networks are still largely unknown. In this study, we used neurons and astrocytes derived from human embryonic stem cells as a cellular model to study the neurotoxicity that is induced by citrate-coated AgNPs (AgSCs). Immunostaining with the astrocyte and neuron markers, glial fibrillary acidic protein and microtubule-associated protein-2 (MAP2), respectively, showed that exposure to AgSCs at the concentration of 0.1 µg/mL increased the astrocyte/neuron ratio. In contrast, a higher concentration of AgSCs (5.0 µg/ml) significantly changed the morphology of astrocytes. These results suggest that astrocytes are sensitive to AgSC exposure and that low concentrations of AgSCs promote astrogenesis. Furthermore, our results showed that AgSCs reduced neurite outgrowth, decreased the expression of postsynaptic density protein 95 and synaptophysin, and induced neurodegeneration in a concentration-dependent manner. Our findings additionally suggest that the expression and phosphorylation status of MAP2 isoforms, as modulated by the activation of the Akt/glycogen synthase kinase-3/caspase-3 signaling pathway, may play an important role in AgSC-mediated neurotoxicity. We also found that AgNO₃ exposure only slightly reduced neurite outgrowth and had little effect on MAP2 expression, suggesting that AgSCs and AgNO₃ have different neuronal toxicity mechanisms. In addition, most of these effects were reduced when the cell culture was co-treated with AgSCs and the antioxidant ascorbic acid, which implies that oxidative stress is the major cause of AgSC-mediated astrocytic/neuronal toxicity and that antioxidants may have a neuroprotective effect.</p

Nanoimaging of Focal Adhesion Dynamics in 3D

<div><p>Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.</p></div

Paxillin localization detected by nSPIRO imaging.

<p>(A) Paxillin-EGFP (pax) and collagen SHG intensity measured by nSPIRO along a cell protrusion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#pone-0099896-g001" target="_blank">Figure 1C</a>. Data were represented as pseudo-images, where the horizontal axis represents intensity along each circular scan and the vertical axis represents the ramp position along the cell protrusion. The non-uniform distribution of paxillin-EGFP was found at both cell protrusion embedded in collagen matrix (800 µm above glass) and closer to glass surface (<250 µm above glass). Furthermore, the position of high paxillin-EGFP intensity is adjacent to collagen fibers, as indicated by black arrows. (B) Nano-imaging of paxillin-EGFP expression on MDA-MB-231 cell protrusion is reconstructed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#s1" target="_blank">introduction</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#pone.0099896.s001" target="_blank">Figure S1</a>. Paxillin-EGFP intensity was color-coded and overlaid with collagen fibers (purple, indicated by white arrows) near the cell protrusion. Paxillin-EGFP shows high intensity spots (yellow arrows) on both sides of the collagen fiber, indicating the cell protrusion may ‘grab’ the collagen fiber. The axis represents 3.2 µm on x, y and z directions. (C) Nano-imaging of paxillin-EGFP expression on U2OS cell protrusion. Similar paxillin distribution as seen from MDA-MB-231 cells can be also seen in U2OS cells. (D) Integrin-EGFP (green) and paxillin-mCherry (red) showed high colocalization (yellow) at MDA-MB-231 cell protrusion, supporting the hypothesis that the paxillin high intensity sites are possible locations of focal adhesions, and the focal adhesions in 3D may be integrin-dependent.</p

Time-lapse images of paxillin-EGFP at cell protrusions.

<p>Time-lapse 3D images from nSPIRO acquisition of an MDA-MB-231 cell protrusion with 15 seconds interval showing the dynamics of paxillin-EGFP. Focal adhesions (high paxillin-EGFP intensity region) were constantly changing the position, as indicated by white arrows.</p

Aggregation and diffusion coefficient differences of paxillin-EGFP and actin-EGFP at FA and non FA.

<p>(A) Schematic diagram and actual image showing the principle of the measurement of paxillin-EGFP dynamics at high intensity region (focal adhesion, FA) and low intensity region (non-focal adhesion, non FA). This measurement provides the aggregation level and diffusion coefficients of the protein. The method was described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#pone-0099896-g001" target="_blank">Figure 1F and 1H</a> as well as in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#s2" target="_blank">Materials and Methods</a> section. (B) Using N&B analysis, paxillin-EGFP brightness measured at FA and non FA locations on MDA-MB-231 cell protrusion were calculated. The brightness, which reflects the aggregation level, of paxillin-EGFP from FA is higher than the brightness from non FA, indicating the presence of paxillin aggregates at focal adhesion in 3D. (C) Although at both FA and non FA locations the free diffusive paxillin can be found, the very slow diffusion population (D<0.03 µm²/s) was identified more frequently in FA location than non FA location for both MDA-MB-231 and U2OS cell lines. (D) Schematic diagram and actual image showing the measurement of actin-EGFP dynamics at cell protrusions with larger radius (∼2 µm, close to the dimension of cell protrusion) and with smaller radius (0.7 µm). (E) MDA-MB-231 cell actin-EGFP brightness value (reflecting the aggregation level) measured with larger and smaller radius do not show significant difference. However, as mentioned in the result, the actin diffusion rate measured was significantly slower than paxillin, indicating the existence of actin polymers or actin binding/unbinding events.</p

Experimental workflow.

<p>(A) First, a raster scan image of an MDA-MB-231 cell with fluorescent expression (green) cultured in type I collagen (red) was taken. In this example, MDA-MB-231 cell was labeled with paxillin-GFP, and the image was taken with 2-photon microscope to detect simultaneously fluorescence and SHG from the collagen. (B) Once the location of cell protrusion was identified, we took zoomed to identify a cell protrusion. Here only the fluorescence image is shown. (C) After identifying the cell protrusion region of interest, we switched the scanning mode from raster scan to nSPIRO. This schematic graph shows the nSPIRO circular scan (yellow) ramps along the cell protrusion (green) identified from (B). The circular scan orbit size was selected according to the cell protrusion radius. While ramping along the cell protrusion, the center position of circular scan is maintained on the cell protrusion by nSPIRO feedback method, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#s2" target="_blank">Materials and Methods</a> section. The intensity profiles of fluorescent proteins in cell protrusion as well as SHG of nearby collagen fibers aree recorded simultaneously. (D) A schematic graph showing the cross section of the protrusion shown in (C) in green, and the trajectory of circular orbit with amplitude modulation (white). The yellow oval shape represents the PSF. The PSF is elongated along z direction, resulting in higher intensity near 90° and 270° due to larger observation volume overlap with the fluorescent structure, and lower intensity near 0° and 180°. (E) After collecting the data from (C), the 3D image reconstruction can be done. The reconstruction utilizes circular scan location and radius to create cylindrical-shaped mesh covered with a texture which represents the intensity profile. (F) Schematic graph showing that the circular scan method can also be applied at a point of interest while the nSPIRO tracking routine maintains the orbit centers on the protrusion. This technique allows the measurement of protein dynamics at a specific orbit location with high temporal resolution. (G) A schematic graph showing the cross section of the protrusion shown in (C) in green, and the trajectory of circular orbit without amplitude modulation (white). The arrow indicates the orbit scan direction, and 0° represents the orbit starting point. The yellow oval shape represents the PSF. The amplitude modulation was not used to avoid introducing artifact for image correlation analysis. (H) Circular scans data from (F) were represented as a pseudo-image in which the horizontal dimension is pixel position along a circular scan, which corresponds to the position 0° to 360° in (G), and the vertical dimension is time. In the pseudo-image, the intensities around 90° and 270° were higher than at 0° and 180° due to the PSF shape, which is different in the x and z directions, as explained in (D). The color scale shows that this effect is not very large, accounting for about 20% variations along the orbit. The data were further analyzed to retrieve protein diffusion coefficients and aggregation level, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099896#s2" target="_blank">Materials and Methods</a>.</p

STICS analysis showing collagen fiber displacement.

<p>(A and B) STICS analysis was applied to a 10 minutes time-lapse movie of collagen SHG signal (purple) and MDA-MB-231 cells with actin-EGFP expression (green) taken by the two-photon microscope to collect the SHG of collagen. (A) Shows the beginning and (B) shows the end of the time-lapse. (C and D) STICS was applied to 64×64 pixel regions on the entire 256×256 pixels image for both channels. (C) Shows STICS analysis on collagen. The yellow box shows the size of the sliding region used for analysis. Arrows indicate the speed and the direction of the average motion at each 32×32 pixel region. The red circle shows a region of collagen fiber that moved toward the opposite side of cell retraction, indicating possible detach and relaxation of collagen fiber from the cell. (D) Shows the SITCS analysis on the fluorescence signal form the cell protrusion, which has more uniform movement.</p

MDA-MB-231 cells in 3D type I collagen matrix.

<p>(A–D) 2-photon excitation raster scan images of MDA-MB-231 cells labeled with paxillin-EGFP (green) in type I collagen matrix (purple) at different depth from the glass surface, as indicated in each panel. Cells on the glass surface show focal adhesions at the cell borders (A), but there are no distinctive focal adhesions when cells are in collagen matrix (B–D, depth is the distance to the glass surface). However, collagen fibers were remodeled near the cells, showing more straight fibers toward the cell body as indicated by the white arrows, and collagen fibers away from cells were more randomly distributed, as shown in panel B. (E) Depth color-coding showing that MDA-MB-231 cells embedded in collagen matrix (750 µm above glass) have protrusions across tens of micrometers in depth, but the major cell bodies were about the same height.</p