Regulation of microtubule dynamics in nerve processes

In this study, we used a combination of electron microscopy, immunocytochemistry, low-light level digital fluorescence microscopy, total internal reflection fluorescence microscopy, and photobleaching to analyze MT dynamics in Xenopus embryo neurons in culture. Neurons were transfected with GFP-tubulin and single fluorescently labeled MTs were imaged at the growth cone and in the axonal shaft of neurites produced by Xenopus embryo neurons. In both regions MTs displayed periods of assembly/disassembly. Quantitative analysis of MT dynamics indicated that parameters of MT dynamic instability are identical for single MTs in the axonal shaft and at the growth cone region.; According to the generally accepted polymer transport model, tubulin is assembled into MTs in the cell body and transported along the axon as a polymer. Since the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. In an attempt to detect this "stop and go" MT transport, we measured translocation of MT plus ends in the axonal shaft by expressing in neurons fluorescently-labeled marker of growing MT plus ends GFP-EB1. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.; To measure MT polarity in growing neurites, we examined translocation of GFPEB1 with time-lapse fluorescence microscopy. Unexpectedly, we found that MT polarity is mixed with ≈18% of MT plus ends directed towards the cell body. Quantitative analysis of MT dynamics suggested that plus end-proximal MTs are stationary. Examination of MT assembly/disassembly at the growth cone region revealed strong coupling between MTs and retrograde actin flow, formation of MT loops, and persistent elongation of plus end-proximal MTs towards the cell body. Our results indicate that modulation of actomyosin activity at the growth cone region is the primary factor dictating MT orientation in growing nerve processes.