40 research outputs found
Ultra-rapid laser protein micropatterning: screening for directed polarization of single neurons
Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.Hertz Foundation (Fellowship)National Institutes of Health (U.S.) (R01 EUREKA Award 1-R01-NS066352)David & Lucile Packard Foundation (Award in Science and Engineering
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Identification of Small Molecules that Enhance Synaptogenesis Using Synapse Microarrays
Synaptic function is affected in many brain diseases and disorders. Technologies for large-scale synapse assays can facilitate identification of drug leads. Here we report a “synapse microarray” technology that enables ultra-sensitive, high-throughput, and quantitative screening of synaptogenesis. Our platform enables the induction of synaptic structures in regular arrays by precise positioning of non-neuronal cells expressing synaptic proteins, while allowing neurites to grow freely around these cells. The technology increases by tenfold the sensitivity of the traditional assays, and simultaneously decreases the time required to capture synaptogenic events by an order of magnitude. It is readily incorporated into multiwell formats compatible with industrial high-throughput screening platforms. Using this technology, we screened a chemical library and identified novel histone deacetylase inhibitors that improve neuroligin-1 induced synaptogenesis via modulating class-I histone deacetylases. We also found a structure-activity relationship for designing novel potent histone deacetylase inhibitors, which can be applied towards development of new therapeutics
Synchronous Symmetry Breaking in Neurons with Different Neurite Counts
As neurons develop, several immature processes (i.e., neurites) grow out of the cell body. Over time, each neuron breaks symmetry when only one of its neurites grows much longer than the rest, becoming an axon. This symmetry breaking is an important step in neurodevelopment, and aberrant symmetry breaking is associated with several neuropsychiatric diseases, including schizophrenia and autism. However, the effects of neurite count in neuronal symmetry breaking have never been studied. Existing models for neuronal polarization disagree: some predict that neurons with more neurites polarize up to several days later than neurons with fewer neurites, while others predict that neurons with different neurite counts polarize synchronously. We experimentally find that neurons with different neurite counts polarize synchronously. We also show that despite the significant differences among the previously proposed models, they all agree with our experimental findings when the expression levels of the proteins responsible for symmetry breaking increase with neurite count. Consistent with these results, we observe that the expression levels of two of these proteins, HRas and shootin1, significantly correlate with neurite count. This coordinated symmetry breaking we observed among neurons with different neurite counts may be important for synchronized polarization of neurons in developing organisms
Conductive-probe atomic force microscopy characterization of silicon nanowire
The electrical conduction properties of lateral and vertical silicon nanowires (SiNWs) were investigated using a conductive-probe atomic force microscopy (AFM). Horizontal SiNWs, which were synthesized by the in-plane solid-liquid-solid technique, are randomly deployed into an undoped hydrogenated amorphous silicon layer. Local current mapping shows that the wires have internal microstructures. The local current-voltage measurements on these horizontal wires reveal a power law behavior indicating several transport regimes based on space-charge limited conduction which can be assisted by traps in the high-bias regime (> 1 V). Vertical phosphorus-doped SiNWs were grown by chemical vapor deposition using a gold catalyst-driving vapor-liquid-solid process on higly n-type silicon substrates. The effect of phosphorus doping on the local contact resistance between the AFM tip and the SiNW was put in evidence, and the SiNWs resistivity was estimated
Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures
We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions
Bright-field micrographs of four different neurons.
<p>Neurite count (<i>N</i>) and polarity (<i>P</i>) using Eq. (1) are also indicated for each neuron. <b>A</b> and <b>B</b> show examples of neurons with two neurites that are relatively more (<b>A</b>) or less (<b>B</b>) polarized. <b>C</b> and <b>D</b> similarly show neurons with many neurites that are relatively more (<b>C</b>) or less (<b>D</b>) polarized. The image in <b>A</b> was taken 28.5 h after plating, and <b>B</b>–<b>D</b> were taken at 52.5 h after plating. All scale bars are 25 µm.</p
HRas and shootin1 expression in developing hippocampal neurons as a function of neurite count after 40 h in culture.
<p><b>A</b>, Typical immunocytochemical stain for HRas in a neuron with 5 neurites. <b>B</b>, Typical immunocytochemical stain for shootin1 in a neuron with 3 neurites. The brightness in both <b>A</b> and <b>B</b> were saturated to make the neurites more visible. When these images were analyzed to determine relative HRas and shootin1 expression levels, image brightness was kept unsaturated. <b>C</b>–<b>D</b>, HRas/shootin1 expression as a function of neurite count. Individual neurons are indicated by plus signs, while the solid line indicates linear fits to the data. In both trend lines, the slope was significantly positive (<i>p</i> < 10<sup>−4</sup> for both fits).</p
Dynamics of neuron polarization as defined by Eq. (1).
<p><b>A</b>, Polarity versus time. The solid line is a linear fit to the data. <b>B</b>, Polarity versus total neurite length. Total neurite length was binned at intervals of 20 µm. The solid line is a Gaussian fit to the data, with an inflection point at 114 µm suggesting a phase transition between unpolarized and polarized states. The nonlinearity of this data agrees with previous work on the polarization of neurons with exactly two neurites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054905#pone.0054905-WissnerGross1" target="_blank">[19]</a>.</p
Experimentally and theoretically predicted symmetry breaking in neurons with different neurite counts.
<p>Each color series indicates a different neurite count, as listed in the legend. <b>A</b>, Experimental measurements of neuronal polarity as a function of time and neurite count. Dashed lines separate data from different discrete time points. Bars indicate mean plus/minus SE, and neurite counts at each time point are only shown if at least 3 neurons had that neurite count at that time point. <b>B</b>–<b>D</b>, Computationally predicted polarity vs. time curves for neurons with different neurite counts using the Samuels (<b>B</b>), Fivaz (<b>C</b>), and Toriyama (<b>D</b>) models. <b>E</b>, Computationally predicted polarity vs. time curves for neurons with different neurite counts using a modified version of the Fivaz model in which HRas expression is independent of neurite count. The original Fivaz model, in which HRas expression increases with neurite count, is shown in <b>C</b>.</p