Growth cone navigation is guided by extrinsic environmental proteins, called guidance cues. Many in vitro studies have characterized growth cone turning up and down gradients of soluble guidance cues. Although previous studies have shown that axonal elongation rates can be regulated by gradients of surface-bound molecules, there are no convincing demonstrations of growth cones turning to migrate up a surface-bound gradient of an adhesive ligand or guidance cue. In order to test this mode of axonal guidance, we used a photo-immobilization technique to create grids and gradients of an adhesive laminin peptide on polystyrene culture dish surfaces. Chick embryo dorsal root ganglia (DRGs) were placed on peptide grid patterns containing surface-bound gradients of the IKVAV-containing peptide. DRG growth cones followed a path of surface-bound peptide to the middle of a perpendicularly oriented gradient with a 25% concentration difference across 30 microm. The majority of growth cones turned and migrated up the gradient, turning until they were oriented directly up the gradient. Growth cones slowed their migration when they encountered the gradient, but growth cone velocity returned to the previous rate after turning up or down the gradient. This resembles in vivo situations where growth cones slow at a choice point before changing the direction of axonal extension. Thus, these results support the hypothesis that mechanisms of axonal guidance include growth cone orientation by gradients of surface-bound adhesive molecules and guidance cues.
A photoimmobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.
Gradients of biological molecules on a microscale have been postulated to elicit cellular responses, such as migration. However, it has been difficult to prepare such gradients for experimental testing. A means for producing such gradients has been developed using a heterobifunctional photolinking agent with laser light activation. The photolinking agent synthesized includes an N-hydroxysuccinimide group and a photoreactive benzophenone (BP) separated by a tetraethylene glycol (TEG) spacer. The presence of the tetraethylene glycol spacer renders the photolinker hydrophilic, a desirable trait for conjugation in aqueous solutions. The linker was then conjugated to R-phycoerythrin (R-PE), a fluorescent protein. The resulting photolinker-R-phycoerythrin conjugate (BP-TEG-PE) was then immobilized onto a polystyrene surface by laser irradiation on a motorized stage. By varying exposure time of the sample to the beam, the amount of BP-TEG-PE immobilized on the surface was changed over an order of magnitude over a distance of 250 microns. This method can be applied to prepare gradients of proteins that elicit biological responses, such as extracellular matrix proteins or growth factors, and to study the biological effects of such gradients.
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