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Site Updated:
10/24/2009

Bio-Engineering Research

Researchers in our lab are actively inventing novel technologies that allow precise manipulation and analysis of cellular activities using non-traditional optical, electrical, thermal, mechanical methods. Currently our research activities are focused in the following three areas.

(1) Neurobiology - We are developing an all optical interface for parallel, remote, and spatiotemporal control of neuronal activities, which will allow us to apply micron-scale sub-cellular stimulation to isolated neurons for in vitro study of activity dependent neuron development, and to apply single neuron stimulation in live animal for in vivo study of neuron network formation.

(2)  Cell biology and tissue engineering - We apply the most advanced micro and nano fabrication techniques to create functional 2D surfaces and 3D micro-structures to study cell behavior in response to different microenvironments.

3) Bio-sensing - Based on our cutting-edge research on nano photonics and metamaterial, our lab is exploring new possibilities to directly visualize and detect cellular activities at molecular level.

Probing Cytoskeleton Dynamics

Background

Cell migration, which involves complicated coordination of cytoskeleton elements and regulatory molecules, plays a central role in a large variety of biological processes from development, immune response to tissue regeneration. However, conventional methods to study in vitro cell migration are often limited to stimulating a cell along a single direction or at a single location. This restriction prevents a deeper understanding of the fundamental mechanisms that control the spatio-temporal dynamics of cytoskeleton.

Methods

In this lab we used a novel microfabricated platform that enables a multi-directional stimulation to a cell using topographical cues. We called TopoSurface. In this device, cells were seeded on a grid-patterned topographical structured surface composed of 2 μm wide and 2 μm high straight ridges. Because the size of a unit grid was smaller than a single cell, each cell was simultaneously experiencing contact guidance leading to different directions.

Results

Figure 1. Cell origami. Reorganization of actin cytoskeleton on a zigzagging patterned TopoSurface, demonstrating that the rigidity of the cytoskeleton does not prevent a normally elongated cell from making sharp turns when the cell is properly guided. Black – phase contrast image of TopoSurface; orange: actin; blue: nucleus. Scale bar, 20 μm.

Figure 2. Multi-directional topographical guidance on grid patterned TopoSurface. (n) normal cells consistently align along the longer side of the grid. (j) Cells treated with Blebbistatin, a myosin II inhibitor, lost control of cell cytoskeleton under multi-directional guidance, extending protrusions along the topographical guiding ridges in all directions. 

Reference paper:

Creating stable surface-bound protein gradient for study of axon initiation and growth cone turning

       Extracellular gradients of secreted guidance factors are known to guide axon pathfinding and neuronal migration. These factors are likely to bind to cell surfaces or extracellular matrix, but whether and how they may act in bound gradients remain largely unclear.

       In this project, we developed a new technique for rapid production of stable
microscopic gradients of substrate-bound proteins. This method uses a removable agarose stamp in contact with an epoxy-coated glass coverslip. The protein solution is first delivered into the miniature source channels prefabricated at the bottom of the agarose stamp. Following a defined duration that allows the establishment of the
intended diffusible protein gradient in the matrix and the printing of proteins onto the substrate surface by covalent bonding, the stamp is removed from the glass substrate and stable bound gradients of protein are obtained. Hundreds of bound protein gradients can be produced in parallel within one operation.

                   Figure 1. Schematic diffusive printing process

       Using this method, we found that bound gradients of netrin-1 and brain-derived neurotrophic factor (BDNF) can polarize the initiation and turning of axons in cultured hippocampal neurons. Furthermore, bound BDNF gradient caused attractive and repulsive polarizing response on gradients of low and high average density of BDNF, respectively. This novel bidirectional response to BDNF depended on the basal level of cAMP in the neuron. Finally, our data showed that the neuron's attractive response to bound BDNF gradient depended on the absolute difference rather than the relative difference in the BDNF density across the neuron, with a minimal effective difference of 1-2 BDNF molecule/μm2 on the substrate surface.

Figure 2. (a,b) Representative fluorescent neuron images showing axon response to bound netrin-1 gradient. (c) Composite tracing of  40 axons on bound netrin-1 gradient. Green arrows represent the direction of increasing density of netrin-1.

       Thus, substrate-bound guidance factors are highly effective in polarizing axon initiation and growth, and the diffusive printing technique is useful for studying neuronal responses induced by bound protein gradients.

Junyu Mai, Lee Fok, Hongfeng Gao, Xiang Zhang, Muming Poo, "Axon Initiation and Growth Cone Turning on Bound Protein Gradients", Journal of Neuroscience, vol. 29, pp 7450, 2009 view pdf