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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.