Abstract
Inhibition of microtubule dynamic instability prevents growth cone turning in response to guidance cues, yet specific changes in microtubule polymerization as growth cones encounter boundaries have not been investigated. In this study, we examined the rate and direction of microtubule polymerization in response to soluble nerve growth factor (NGF) and immobilized chondroitin sulfate proteoglycans (CSPGs) by expressing enhanced GFPβEB3 in rat pheochromocytoma (PC12) cells. GFPβEB3 comets were monitored in live cells using timeβlapse epifluorescent microscopy. With an automated tracking system, the rate of microtubule polymerization was calculated as the frameβtoβframe displacement of EB3 comets. Our results demonstrate that the rate of microtubule polymerization is increased following NGF treatment, whereas contact with CSPGs decreases microtubule polymerization rates. This reduction in microtubule polymerization rates was specifically localized to neurites in direct contact with CSPGs and not at noncontacting neurites. Additionally, we found an increase in the percentage of microtubules polymerizing in the retrograde direction in neurites at CSPG boundaries, with a concomitant decrease in the rate of retrograde microtubule polymerization. These results implicate localized changes in microtubule dynamics as an important component of the growth cone response to guidance cues. Β© 2010 WileyβLiss, Inc.