Caroline E Kingman | PubMed Health

PubMed2006

Effects of substrate geometry on growth cone behavior and axon branching.

At the leading edge of a growing axon, the growth cone determines the path the axon takes and also plays a role in the formation of branches, decisions that are regulated by a complex array of chemical signals. Here, we used microfabrication technology to determine whether differences in substrate geometry, independent of changes in substrate chemistry, can modulate growth cone motility and branching, by patterning a polylysine grid of narrow (2 or 5 microm wide) intersecting lines. The shape of the intersections varied from circular nodes 15 microm in diameter to simple crossed lines (nodeless intersections). Time-lapse recordings of cultured hippocampal neurons showed that simple variations in substrate geometry changed growth cone shape, and altered the rate of growth and the probability of branching. When crossing onto a node intersection the growth cone paused, often for hours, and microtubules appeared to defasciculate. Once beyond the node, filopodia and lamellipodia persisted at that site, sometimes forming a collateral branch. At nodeless intersections, the growth cone passed through with minimal hesitation, often becoming divided into separate areas of motility that led to the growth of separate branches. When several lines intersected at a common point, growth cones sometimes split into several subdivisions, resulting in the emergence of as many as five branches. Such experiments revealed an intrinsic preference for branches to form at angles less than 90 degrees . These data show that simple changes in the geometry of a chemically homogeneous substrate are detected by the growth cone and can regulate axonal growth and the formation of branches.

Animals↗

PubMed2003

Patterning axonal guidance molecules using a novel strategy for microcontact printing.

We present here a two-step strategy for micropatterning proteins on a substrate to control neurite growth in culture. First, conventional microcontact printing is used to prepare a micropattern of protein A, which binds the Fc fragment of immunoglobulins. Then, a chimeric protein, consisting of the extracellular domain of a guidance protein recombinantly linked to the Fc fragment of IgG (prepared using conventional molecular techniques), is applied from solution. The chimeric protein binds to the patterned protein A, taking on its geometric pattern. Using this method, we have micropatterned the extracellular domain of the cell adhesion molecule, L1 (as an L1-Fc chimera) and demonstrated that it retains its ability to selectively guide axonal growth. L1-Fc micropatterned on a background of poly-L-lysine resulted in selective growth of the axons on the micropattern, whereas the somata and dendrites were unresponsive. Substrates bearing simultaneous micropatterns of L1-Fc and poly-L-lysine on a background of untreated glass were also created. Using this approach, cell body position was controlled by manipulating the dimensions of the poly-L-lysine pattern, and the dendrites were constrained to the poly-L-lysine pattern, while the axons grew preferentially on L1-Fc. The two-step microcontact printing method allows preparation of substrates that contain guidance proteins in geometric patterns with resolution of approximately 1 microm. This method should be broadly applicable to many classes of proteins.

Axons↗