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One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes

The stability and deformability of the cell nucleus are important to many biological processes like migration, proliferation, polarization. When cells are exposed to mechanical force, the force will be transmitted via cytoskeleton to the nucleus, induce shape deformation of the nuclear envelopes, and even change the configurations of nucleoskeletons. However, current techniques for studying nuclear mechanics are limited for studying inducing the effects of subcellular force perturbation in live cells. Here we developed a novel assay of using vertical nanopillar arrays to study the mechanical coupling between cell nucleus and cytoskeleton in live cells. Our results showed that nanopillars can induce deformation of nuclear envelope. By changing the geometry of the nanopillars or the stiffness of the nucleus, we can control the degree of nuclear deformation. Also, cytoskeletons such as actin and intermediate filaments were showed to play important roles in inducing and preventing nuclear deformation, respectively. Furthermore, we showed that mechanical perturbation of the nuclear envelope can cause the reorganization of nuclear lamina, which give the clue that cell nucleus itself may be able to sense and respond to mechanical signals. Overall, vertical nanopillars provide a long-term and non-invasive force to create a subcellular nuclear perturbation, and can be used as a tool for studying nuclear mechanotransduction in live cells.

The gold standard for investigating neuronal electrical activities has been electrodes. Modern electrophysiology methods boast high sensitivity and temporal resolution with the ability to measure single ion channels and networks of neurons. However, the spatial resolution and robustness are limited by the size and geometry of the electrode. In this era, optical methods have picked up momentum as powerful tools to measure the electrical activities of neurons by using an optical probe that transduces the electrical signal into an optical signal.Prussian blue (PB) is a mixed-valence inorganic material composed of alternating ferric and ferrous ions in a cubic, cyano-bridged lattice with open sites for alkaline metal intercalation. This material is known for its biocompatibility, insolubility in water, activities toward alkaline metal ions and electrochromicity. It is the latter property that makes Prussian blue an excellent candidate to measure the electrical potentials of excitable membranes. The color of this material depends on the applied electrical potential which in turn controls the redox state of the irons. Its blue color is turned to colorless by reduction and is recovered by oxidation. We hypothesize that the electrical potential of excitable membranes will modulate the spectral properties of the material which will be detected through a differential photodiode detector.A PB thin film was electrodeposited onto an ITO-coated glass slide and its optical properties were characterized. As a model for electrophysiology measurements, a clone line of modified HEK 293 cells that stably express Nav 1.3 and KIR 2.1 and generate spontaneous electrical action potentials were used. Herein, we demonstrate the ability to detect the extracellular action potentials of HEK 293 cells and evaluate the film's potential for imaging networks of neuronal cells.

Despite remarkable advances in characterizing molecular motors and microtubular transport in vitro, our understanding of intracellular cargo transport is rudimentary. The intracellular mechanochemical properties and cooperative mechanics of multiple motors, sharing load and coordinating cargo direction, are fundamental in this regard. To elucidate these, we developed two novel approaches for manipulating the transport of axonal endosomes, loaded with nanoprobes (

Nanotechnology innovations have advanced biological science by providing new tools for probing cellular process. Whether or how cellular processes are altered upon interacting with such small scale devices are, however, not well understood. One crucial yet overlooked phenomenon is that nanostructures can induce local curvatures on plasma membrane. Modulation of local membrane curvature is known to be important in creating micro-domains for endocytosis initiation.In the present work, we used electron-beam lithography to make patterned nanopillars arrays with controllable diameters from 60nm to 2000 nm. From both SEM and TEM studies, nanopillars were found to sufficiently induce membrane curvature in living cells. By culturing mammalian cell lines with fluorescent protein labeled clathrin and dynamin on nanopillar substrates, recruitment of these two key proteins in endocytosis machinery were found to preferentially happen on nanopillars in comparison to flat surfaces. Similar phenomenon was also found in adaptor protein AP2 and BAR domain proteins. More interestingly, when changing the nanopillar to other geometries, e.g. nanobar and nanoCUI, such recruitment was found to more correlated with positive and large curvatures. We further studied the dynamics of clathrin and dynamin on nanostructures with gradient geometry, and differential effects were observed. This work provides new insights on the curvature dependent recruitment of endocytosis machinery proteins in living cells, and demonstrates the possibility of using nanofabricated structures as a new platform for membrane curvature manipulation.

Three-dimensional (3D) nanoelectrodes fabricated on standard multielectrode array architecture have proven useful in monitoring intracellular electrophysiology of cardiomyocytes. The electrodes’ mechanism of action involves a voltage pulse at the electrode which exceeds the dielectric breakdown of an engulfing cell's membrane, electroporating the cell and dramatically reducing the impedance to intracellular potential recording. The electroporation mechanism is non-invasive as the pores reseal after such recording events. This gives rise to a method which is useful in its ability to multiplex, its technical ease of use, and non-invasiveness.The driving phenomenon which gives this technological gain over planar electrodes is an enhancement of the seal resistance between the electrogenic center (the cell) and the recording center (the nanoelectrodes). Cells in vitro engulf 3D electrodes such that the membrane-electrode distance is much reduced compared to that of the membrane-planar electrode distance. This membrane-electrode distance is dependent upon the geometry of the electrode, but the electrical ramifications of this have not been characterized to date. Herein we explore this dependence by using electrochemical impedance spectroscopy in correlation with reduced-artifact FIB/SEM and electrophysiological measurement of HL-1 cardiomyocytes over an order of magnitude in 3D electrode diameter.

Recent years have seen numerous applications of nanoelectronic devices for cell electrophysiology measurements. Here we present vertically aligned Pt and Au nanopillar arrays for both extracellular and intracellular recording of HL-1 cardiomyocytes. The small footprint of our nanopillar arrays holds the advantage of high spatial resolution recording. We discover that the tight cell membrane-electrode interface allows recording of a large extracellular signal despite the small detection area. After local electroporation of the cell membrane around the pillars, we demonstrate intracellular recording of action potentials. Because this method is minimally invasive, we are able to record action potentials from the same cell over a span of three days.

Interfacing cells with micro- and nano-electronic devices has been intensively studied over the last decade. However, a long-term and efficient electrical cell interface is yet to be accomplished. Here we report that vertically aligned nanoscale electrode arrays, which promote tight attachment to cell membrane, form good electrical coupling with cultured cardiomyocytes and neuron cells. Scanning electron microscopy (SEM) analysis shows that cells readily engulf nano-electrodes by wrapping around them. The tight junction between cells and nano-electrodes enables high quality and long-term electrical cell interface, which allows us to achieve non-destructive action potential recording with intercellular-like signal quality.View Large Image | View Hi-Res Image | Download PowerPoint Slide

It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to induce different cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of extracellular signal-regulated kinase (ERK) and result in the distinct outcomes of differentiation and proliferation, respectively. However, direct and quantitative linkage between time kinetics of ERK activation and the cellular response is still lacking due to difficulties in perturbing the kinetics of intracellular signaling pathways. Here, we construct a light-gated protein-protein interaction system that uses light to regulate the activation and inactivation of ERK. We find that light-induced ERK activation alone is sufficient to stimulate significant neurite outgrowth in PC12 cells in the absence of growth factors. Intermittent on/off light control reveals a memory effect in ERK-stimulated neurite outgrowth in PC12 cells. The memory effect shows a 45 min off-time threshold, below which a full-speed neurite outgrowth is maintained despite that ERK is gradually turned off. When the off-time is greater than the threshold, the speed of neurite outgrowth decreases with a half time of 2 h as cells slowly lose their memory of prior ERK activation. Interestingly, the 45-min time threshold and the 2-h half time memory are independent of the prior duration of ERK activation. Overall, light-controlled signaling kinetics enables precise dissection of the temporal dimension of signal transduction in cells.

Axons of neurons present a unique challenge for intracellular transport: with a diameter of roughly one micrometer and length that can range up to a meter, transport machinery in the axon must be able to move cargo processively with high speed while moving largely unidirectionally. Motile cargoes must also be able to bypass static organelles which can be hundreds of nanometers in size, taking up significant portions of the cross section of the axon. Defects in this transport are found in a host of neurodegenerative disorders, including Alzheimer's and Parkinson's Diseases. One strategy that cells use to maintain healthy axonal transport is to provide each cargo with several copies of different molecular motors. However, details of the regulation required for appropriate teamwork between the motors are lacking. The motion that results from the actions of multiple motors moving a single cargo inside the cell can be complex, and conventional imaging approaches are limited to measuring the position of cargo along the length of the axon. We present an experimental approach to measure an additional parameter - cargo orientation. By constructing a dual-polarization dark field microscope, we achieve a high throughput readout of position and orientation of gold nanorod-containing endosomes in primary neurons with millisecond resolution. This allows us to relate particular translational-orientational behaviors specifically to teams of either kinesins or dyneins. We observed that changes in cargo velocity correlate with changes in orientation particularly at transitions between paused and moving states. Furthermore, we find that cargoes with similar translational dynamics can have very different orientational behavior and individual cargoes, though of identical origin, differ in global orientational dynamics.

Actin filament is an essential component of the cell cytoskeletal system under the physiological conditions. In addition to their roles in supporting cell shape, actin filaments act as molecular tracks for myosin motors that are involved in the movement of organelles such as mitochondria in the axon. However, how actin filaments regulate axonal transport processes are yet to be fully elucidated.Nerve growth factor binds and activates its receptor located at axon terminus, which intriguers the complex to be endocytosed sorted into signaling endosomes. NGF-containing endosomes are retrogradely transported from the axon terminus to the cell body. In this study, we investigated the effects of actin filiments on axonal transport by tracking the transport of single NGF modified with the quantum dot using microfluidic device.Embryonic DRG neurons were cultured in the microfluidic nerve cell chambers made by PDMS. The microfluidic chamber allows us to apply latrunculin B, an actin de-polymerization inhibitor, exclusively to the middle segment of axon. This treatment would not affect signaling processes in the cell body or the endocytosis process that happens at the axon termini. We monitored the retrograde transport of Qdot-NGF in actin-depleted axons using TIRF microscopy. We found that NGF axonal transport continues in axons that are depleted of actin filiments, confirming previous reports that NGF transport is a microtubule-based process. However, we found that the average speed of axonal transport slowed down in Latrunculin B treated axons. Detailed analysis of why actin depolymerization affects axonal transport is still in progress.

Effective electronic neuron interface requires tight attachment between electronic devices and neuron cells. However, it is the nature of living cells to maintain an extracellular cleft between their membrane and the substrate which they adhere to, and the cleft contribute to most of the signal leakage. Nano-scale electrodes could enable tight attachment to cell membrane and thus form good electrical coupling with cultured neuron cells. We fabricate nano-electrodes by focused ion beam (FIB) Pt deposition. The electrodes are vertical aligned, and with diameter of 200 nm and height of 1 um. We cultured cortical neurons on the substrates with nanoelectrode arrays. Scanning electron microscopy (SEM) analysis shows that neuron cells engulf nano-electrodes readily. The tight engulfment provides good electrical coupling between neuron cells and nano-electrodes. We also examine action potential recording and stimulation with nano-electrode arrays. Image 1 shows SEM images of neuron nano-electrodes interface. The cross section view of the interface is milled by FIB.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The rapidly evolving field of nanotechnology creates new frontiers for biological sciences. Recently, we and other groups show that vertical nanopillars protruding from a flat surface support cell survival and can be used as subcellular sensors to probe biological processes in live cells. In particular, we are exploring nanopillars as electric sensor, optical sensors, and structural probes. As an electric sensor, nanopillars electrodes offer several advantages such as high sensitivity, subcellular spatial resolution, and precise control of the sensor geometry. A sensitive measurement of cellular electrical activities requires strong coupling between the cell membrane and the recording electrodes. We found that nanopillars electrodes deform the cell membrane inwards and induce negative curvature when the cell engulfs them, leading to a reduction of the membrane-electrode gap distance and a higher sealing resistance. The 3D topology of the nanopillars electrodes is crucial for its enhanced signal detection. A new approach explores nanoelectrodes of a new 3D geometry, namely nanotubes with hollow centers. The nanotube geometry further enhances membrane-electrode coupling efficiency and also significantly increases the time duration of intracellular access. Interestingly, nanopillars serve as focal adhesion points for cell attachment. The presence of high membrane curvature induced by vertical nanopillars or nanotubes affects the distribution of curvature-sensitive proteins. Those studies show a strong interplay between biological cells and nano-sized sensors, which is an essential consideration for future development of interfacing devices.

As knowledge of the bulk behavior of biological systems continues to grow, there is an increasing demand for knowledge of cellular processes at the single-molecule level. This presents a unique challenge, a combination of the dynamic nature of the system and the inability to modulate the concentration of the target species. A significant limitation of previous low-penetration methods arises from the very character that provides their utility: the low penetration depth also means they can only probe molecular events very close to the substrate surface. We have fabricated vertical silicon dioxide nanopillars which, at a height of up to one micron, carry that low penetration depth up into the cell environment where the relevant molecular processes occur. The pillars can also be specifically functionalized with molecules of interest for either delivery into the local environment or study while tethered in the observation volume. With single molecule detection at biologically-relevant concentrations and biologically-applicable locations, these nanopillars provide a template on which to study a multitude of biological processes in a controlled, dynamic, and localized fashion.

Retrograde neurotrophic signals, from the axon terminal to the cell body, are essential for the survival and function of neurons. Axonal microtubules serve as polarized tracks for molecular motor proteins driving the signaling endosomes from the axon terminal to the cell body. The robustness of this long-distance transport and the direction specificity can be attributed to the cooperative mechanics of multiple motors and/or specific coordinators in vivo. Noninvasive external force control of axonal endosomes in live neurons is a challenging prospect, which can unravel the transport machinery and the direction regulation mechanisms in vivo. Here, we present an integrated methodology based on microfluidic neuron culture, high-gradient magnetic trapping and pseudo-TIRF imaging that permits external control of axonal endosome transport in live neurons via magnetic forces. We fabricated a novel microfluidic device for neuron culture by patterned electrodeposition of soft micromagnets on glass coverslips. In the presence of an external magnetizing field, the soft micromagnetic pattern gives rise to local zones of high magnetic gradients. By culturing neurons in this device, with axons aligned along these high gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (

Charcot-Marie-Tooth type 2B (CMT-2B) is a neurodegenerative disease characterized by terminal axonal death. Genetic analysis from human CMT-2B patients revealed four missense point mutations (L129F, K157N, N161T, V162M) in their genes encoding a small GTPase Rab7, a marker for late endosomes in the degradation pathway. The exact mechanism of how Rab7 mutants cause CMT-2B remains poorly understood. Here, we analyzed the effect of Rab7 mutants on the signaling and axonal transport of a nerve growth factor (NGF) receptor - TrkA. Fluorescent protein-engineered Rab7 and TrkA were transfected in rat embryonic dorsal root ganglia neuronal cells. Axonal transport of Rab7- and TrkA-containing endosomes was followed by time-stamped live cell fluorescence microscopy. We found that TrkA moves roughly twice as fast as Rab7s, among which CMT-2B associated Rab7 mutants outpace wt-Rab7. Curiously, endosomes co-transfected with both Rab7 and TrkA move even slower than those with singly transfected Rab7. Western blot analysis from Rab7/TrkA-cotransfected PC12 cells showed that the level of phosphorylated TrkA is lower in Rab7 mutants that that in wt-Rab7. Our results suggested that Rab7 mutants can potentially contribute to CMT-2B by dis-regulating NGF-TrkA signaling via perturbing the balance between retrograde and anterograde axonal transport processes. These results imply that axonal transport can be a potential treatment target for CMT-2B neurodegeneration.

The axon acts as a conduit for organized transport of material, between the cell body and the synapse, which is essential for the function and survival of neurons. Axonal traffic jams caused by local accumulation of cargo have been implicated in many neurodegenerative diseases. In order to study the neuronal response to axonal traffic jams we need new noninvasive assays capable of A) slowing/stalling axonal cargo by external forces to induce controlled traffic jams B) monitoring the perturbed transport and the ensuing neuronal response in real time. Here, we present an integrated methodology based on microfluidic neuron culture, high-gradient magnetic trapping and multi-color TIRF imaging that permits external control of axonal traffic in live neurons via magnetic forces. We fabricated a novel microfluidic device for neuron culture by patterned electrodeposition of soft micromagnets (permalloy) on glass coverslips. In the presence of an external magnetizing field, the soft micromagnetic pattern gives rise to local zones of high magnetic gradients. By culturing neurons in this device, with axons aligned along these high gradient zones, we can exert pN forces on axonal endosomes carrying magnetic nanoparticles (MNPs, 50 nm). The magnetic forces counter the molecular motor forces to physically stall the endosomes, which leads to axonal traffic jams. The axonal growth and the delivery of MNP-loaded axonal endosomes along the high gradient zones are achieved by microfluidic compartmentalization of neuron culture. We have successfully A) compartmentalized DRG neurons in prototype magnetic devices B) characterized lectin-mediated axonal transport of 50 nm MNPs by pseudo-TIRF imaging, with/without external magnetic forces C) demonstrated the magnetic induction of controlled axonal traffic jams. These advances can potentially unravel the cooperative mechanics of multi-motor axonal transport and also elucidate the generic links between traffic jams, axonal swellings and neurodegeneration.

Molecular photoswitches can be employed for the study of protein trafficking in living cells and applications in optical memories. Especially, to switch fluorescence, fluorescence quenching mechanism via energy or electron transfer is one of the most fundamental pathways to realize the system of photoswitching. In order to achieve fluorescence photoswitching, photochromic compounds such as diarylethene have been used to toggle fluorescence on and off. For example, photochromic diaryethene induces absorption changes upon light irradiations via cyclization reaction, which would trigger the fluorescence toggling. On the other hand, polymer dots (P-dots) is one of the promising fluorescent probes for the biological applications. We assumed that doping diarylethene into P-dots would realize fabrication of photoswhitchable P-dots via energy transfer mechanism between fluorescent polymer and diarylethene. In this study, we synthesized photoswitchable P-dots doped with diarylethene to toggle the fluorescence back and forth via energy transfer mechanism. We also tried to apply synthesized photoswitchable P-dots toward biological imaging. First, we examined the photoswitching properties with absorption and fluorescence measurements. Fluorescence of P-dots was dramatically quenched upon photoirradiation with UV light and recovered after visible light irradiation. Those photoswitching processes were reversible and could go through at least 5 cycles. We are now applying photoswitchable P-dots synthesized as mentioned above to biological imaging. Details will be discussed at the meeting.

With unique properties and access to length scales pertinent to biological activities, nanoscale structures and materials stand to make significant contributions to the investigation of cell processes. We investigated cellular interactions with vertically-aligned nanopillars of several materials, and the interface between the cells and said vertical nanopillars. Cells exhibit significantly decreased motility across a nanopillar surface as compared with a flat surface, with average movements over a five day period decreased from 57.8um to 3.0um. Additionally, scanning and transmission electron microscopy analyses show tight seals of around 10 nanometers between the cell membrane and nanopillars, in contrast with the tent-like gaps of 100nm-1um typical between cells and flat surfaces. Not only do cells fail to migrate away from nanopillar surfaces, we have also shown that the nanopillars serve to encourage attachment by cell outgrowths and stimulate the axon growth cone in neurons. As such, patterns of nanopillars serve as effective axon-guiding instruments, and can form the basis of templates for the long-term study of neural networks.

Charcot-Marie-Tooth (CMT) neuropathy, characterized by severe sensory neuron loss, is the most common inherited disorder of the peripheral nervous system. Several GTPase Rab7 protein mutants, mainly targeted to the highly conserved amino acid, have been identified in CMT type 2B. Exact mechanism of how such point mutations cause malfunction of neurons is not well understood. Here, we studied how those Rab7 mutations affect their axonal transport in primary rat dorsal root ganglia neurons. Real time fluorescence imaging revealed that Rab7-containing endosomes engage in bi-directional transport in axons, similar to that of TrkA receptors in the same culture. However, the speed of Rab7 transport is significantly slower than that of TrkA. In addition, there is a clear variation in the speed of axonal transport between wild-type Rab7 and mutated Rab7 proteins. Our work suggested that point mutations of Rab7 proteins could potentially cause or contribute to CMT2B neurodegenerative disease by regulating its axonal transport process.

We present a detailed motion analysis of retrograde nerve growth factor (NGF) endosomes in axons to show that mechanical tugs-of-war and intracellular motor regulation are complimentary features of the near-unidirectional endosome directionality. We used quantum dots to fluorescently label NGF and acquired trajectories of retrograde quantum-dot-NGF-endosomes with

The rapidly evolving field of nanotechnology creates new frontiers for biological sciences such as quantum dots for fluorescence imaging and nanotransistor-based biosensors. Recently, vertical nanopillars protruding from a flat surface has been shown to support cell survival and deliver large molecules into the attached cells. Here we demonstrate (1) the use of vertically aligned SiO2 nanopillars to achieve below-the-diffraction limit observation volume in vitro and inside live cells and (2) the use of vertical Pt nanopillars to enhance electrical coupling between neuron cells and the recording electrodes. Transparent SiO2 nanopillars embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates high-localized illumination that can be used for single molecule detection with high fluorescence background. We also fabricate vertically aligned Pt nanopillars to enhance the electrical coupling between cultured neurons and the measuring microelectrode arrays. Pt nanopillars were found to serve as geometrically better focal adhesion points for cell attachment and thus enable high-quality signal recording. Therefore, vertical nanopillars can serve as a versatile platform to optically, electrically and chemically probe biological activities in live cells.

Retrograde transport of nerve growth factor signaling endosomes by microtubular motors, from the axon terminals to cell bodies, is vital for the survival of neurons. The robustness of this fast long-distance axonal transport and biased directionality could be attributed to the cooperative mechanics of multiple motors and/or intracellular regulation mechanisms. Here, we present a comprehensive motion analysis of retrograde nerve growth factor (NGF)-endosome trajectories in axons to show that cooperative motor mechanics and intracellular motor regulation are both important factors determining the endosome directionality. We used quantum dot (QD) to fluorescently label NGF and acquired trajectories of retrograde QD-NGF-endosomes with < 20 nm accuracy at 32 Hz, using pseudo-total internal reflection fluorescence imaging. Using a combination of transient motion analysis and Bayesian parsing, we segregated the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses and brief anterograde reversals. Mean square displacement analysis and the temperature dependence of transient reversals confirm that motors of opposite polarities (dyneins and kinesins) are both active on the endosomes during retrograde transport. Stochastic multi-motor model simulations show that the biased directionality as well as several statistical metrics of NGF-endosome transport can only be simulated reasonably by assuming that the microtubule-binding affinity of kinesin is down-regulated. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 4-7 active dyneins and 1-3 down-regulated kinesins. These observations are corroborated by the dynamics of endosomes detaching under load in axons; showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the ramifications of our results for intracellular transport regulation, in conjunction with recent studies on cellular cargo in a wide range of motility (bidirectional to unidirectional) regimes.

While it can generally be said that cellular function is critically dependent on the fidelity of cargo transport, processive transport is even more important in the axons and dendrites of neurons, where a cell must regulate populations of molecules on length scales that can range up to meters. Consequently, much effort has been made to investigate the translocation of cargoes in neurons and the properties of the motors responsible therein. Though biocompatible fluorophores have become increasingly powerful tools for study of motor-driven transport, they suffer from photobleaching and require bright illumination which can be toxic to live cells. Most conventional fluorescent approaches are further limited by the lack of oreintation information they provide. On the other hand, with the small diameter of neurites and the high levels of traffic they support through a crowded environment, orientation of the cargoes relative to the cytoskeletal tracks they are moving on can be vital. We present an experimental approach making use of dark field optical microscopy and gold nanorods as reporters of both lateral translocation as well as orientation of cargo in neurons. using relatively low illumination intensity, we can measure dynamics of single cargoes moving in the image plane and resolve changes in the azimuth and polar angles all at millisecond time scales. Furthermore, the gold nanorods can be specifically delivered to the cell body or axon terminal by culturing the neurons in microfluidic devices with separate chambers, enabling the investigator to resolve differences between retrograde and anterograde transport. The ability to track axonal transport with a high temporal and spatial dynamic range reveals several kinds of orientational changes of moving cargoes that correlate with transport dynamics, allowing more detailed inferences into changes in the activity of molecular motors.

The Mitogen-Activated Protein Kinase (MAPK) signaling pathway regulates critical cellular function such as cell proliferation, differentiation, and apoptosis. Defective MAPK signaling has been frequently discovered in various cancers such as in breast cancer, lung cancer, and melanoma. Evidence showed that signaling output of the MAPK pathway depends critically on its spatiotemporal regulation. However, there are very limited means to control its spatial and temporal dimension in live cells with high accuracy. Here, we report a light-gated protein-protein interaction system that precisely regulates the activation and inactivation of the MAPK signaling pathway. We show that sustained MAPK activation through continuous light stimulation is sufficient to induce significant neurite outgrowth in PC12 cells in the absence of nerve growth factor. Light-gated activation leads to an interesting discovery that MAPK alone is sufficient to account for neurite elongation, it only partially contributes to the full development of sodium channels in PC12 cells. The strategy of using light-gated protein interaction shows a great promise in dissecting detailed mechanisms of signal transduction in cells.

The structure of the nuclear envelope is crucial to many cell processes, from signal transduction to migration and motility, yet few studies have been carried out about the mechanical properties of the nuclear envelope. Previous studies rely on invasive techniques like micropipette aspiration that require isolation of the nucleus from the cell and neglect the participation of the cytoskeleton and extracellular membrane. In order to fully understand this process, we need a technique that can measure the response of the nucleus in intact cells as they function normally. For this purpose, we fabricated arrays of vertical nanopillars with various diameters, heights, and spacing to observe the extent of both plasma membrane deformation and nuclear deformation in response to non-invasive mechanical perturbation by vertical nanopillars. We found that while the plasma membrane is very flexible and conforms to the shape of the nanopillars, the nuclear membrane is much more rigid and deforms to a lesser degree. All three of those geometric parameters determine the stress relayed to the nucleus by the cell membrane and cytoskeleton. We investigated the contributions of different components of the cytoskeleton and found that while actin pulls the nucleus toward the cell membrane, intermediate filaments resist nuclear deformation. Nanostructures provide a new and exciting platform for studying cell and nuclear mechanics in length scales and contexts that are inaccessible to traditional techniques.

Brain-derived neurotrophic factor (BDNF) is a protein that regulates neuronal survival and synaptic plasticity in brain. BDNF binds and activates receptor tyrosine kinase TrkB and the trafficking of phosphorylated TrkB triggers multiple intracellular pathways involved in neuronal development. It is not clear, however, whether BDNF is transported with TrkB in a signaling complex.It has been reported that the number of surface TrkB and the phosporylation of TrkB is enhanced by high frequency neuronal activity, but whether the trafficking of TrkB is also modulated by neuronal activity has not been addressed. To investigate this problem, we transfect hippocampal neurons with BDNF-eGFP or TrkB-mCherry and separately plate them on two sides of a PDMS chamber. We look at 1) whether BDNF and TrkB are co-transported; 2) whether the transportation flux, speed and other features are affected by high frequency field stimulation. The results may help us to understand the mechanism under synaptic plasticity and memory formation.

Brain derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays important roles in neuron survival, development and synaptic efficacy. It is believed that exogenous BDNF binding to TrkB activates three major signaling pathways: MAPK, PI3K and PLC-gamma. One important issue under these regulation mechanisms is whether and how BDNF is transported into neurons and its intracellular translocation. Furthermore, whether such exogenous BDNF is released and taken up by another neuron and involved in neuron to neuron communication has not been studied. In this work, BDNF conjugated to quantum dot is traced after it is taken by hippocampal neurons. A compartmentalized microfluidic device has been designed to separate axons and dendrites from each other. By applying Qdot-BDNF only to the axon chamber and observing some Qdot-BDNF leaving from the dendrite chamber, it is clearly proved that BDNF up-taken from the axon terminal can be translocated all the way to dendrites. Qdot-BDNF entering and leaving the soma has also been directly observed. Whether BDNF is released from the axon or dendrite terminal is under investigation. It is hoped that at the end of this work, an overall picture of the whole regulation cycle of exogenous BDNF and whether its role as a chemical communicator through the neuronal network can be clearly shown.

During the development of a neuron cell, multiple dendrites and a single axon, which have molecularly and functionally distinct domains, will generate from the soma to enable the directional communication between cells. The initiation and extension of such structures are critical to neuron development and neural circuit formation. In vitro neuron culture system has been a major model to study axon initiation and elongation, and many regulating genes have been identified in the in vitro model. However, only a few of the genes have been proven to be required in vivo, which may mainly due to the lack of extracellular cues in the in vitro model. The importance of extracellular cues to axon initiation and outgrowth is therefore emerging as a major theme in neural development. Nanostructures and nanomaterials serve as promising candidates to provide topographical cues to neuronal adhesion and development. Previous studies showed that neuron cells were able to sense nanoscale structures and responded differently in their neurite extension. In the present work, we use patterned nanopillar structures as controllable topographical cues to culture hippocampal neurons, and found that nanopillars have significant guidance effect on neurite outgrowth and elongation. More interestingly, the axon specification occurs in the first 12 hours after cell plating, which is much earlier than the usual time point for cells growing on normal flat surfaces. It indicated that the topographical cues can indeed accelerating neural development. We further studied this topographical influence on axon initiation and elongation by varying the diameter, height, pitch and shape of the nanopillars, and different effects were observed. This work will provide new insights on the role of topographical cues for neuronal development in vivo, as well as the possibility of using nanoscale topographic features to control neuronal development.