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Broadie Laboratory

In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila.

AUTHORS

Yan YYanping , Broadie K Kendal . Journal of neuroscience methods. 2007 5 15; 162(1-2). 198-205

ABSTRACT

Disrupted microtubule dynamics in neuronal synapses has been suggested as an underlying cause for several devastating neurological diseases, including Hereditary Spastic Paraplegia (HSP) and Fragile X Syndrome (FXS). However, previous studies have been restricted to indirect assays of synaptic microtubules, i.e. immunocytochemistry of microtubule-associated proteins and post-translationally modified tubulins characteristic of microtubules with different stabilities. Very little is known about synaptic microtubule dynamics in vivo, or how microtubule dynamics may be disrupted in disease states. In this study, we develop methods to analyze microtubule dynamics directly in living synaptic boutons in situ. We use fluorescence recovery after photobleaching (FRAP) of transgenic green fluorescent protein (GFP) tagged tubulin at the well-characterized Drosophila neuromuscular junction (NMJ) synapse. FRAP measurements of tubulin-GFP demonstrate biphasic recovery kinetics. Treatment with taxol to stabilize microtubules and promote microtubule assembly reduces both recovery phases. Treatment with vinblastine to disassemble microtubules increases the fast recovery phase and decreases the slow recovery phase. These data indicate that the fast recovery phase is generated by rapid diffusion of tubulin subunits and the slow phase is generated by the relatively slow turnover of microtubules. This study demonstrates that tubulin-GFP fluorescence recovery after photobleaching can be used to assay microtubule dynamics directly in living synapses.

Disrupted microtubule dynamics in neuronal synapses has been suggested as an underlying cause for several devastating neurological diseases, including Hereditary Spastic Paraplegia (HSP) and Fragile X Syndrome (FXS). However, previous studies have been restricted to indirect assays of synaptic microtubules, i.e. immunocytochemistry of microtubule-associated proteins and post-translationally modified tubulins characteristic of microtubules with different stabilities. Very little is known about synaptic microtubule dynamics in vivo, or how microtubule dynamics may be disrupted in disease states. In this study, we develop methods to analyze microtubule dynamics directly in living synaptic boutons in situ. We use fluorescence recovery after photobleaching (FRAP) of transgenic green fluorescent protein (GFP) tagged tubulin at the well-characterized Drosophila neuromuscular junction (NMJ) synapse. FRAP measurements of tubulin-GFP demonstrate biphasic recovery kinetics. Treatment with taxol to stabilize microtubules and promote microtubule assembly reduces both recovery phases. Treatment with vinblastine to disassemble microtubules increases the fast recovery phase and decreases the slow recovery phase. These data indicate that the fast recovery phase is generated by rapid diffusion of tubulin subunits and the slow phase is generated by the relatively slow turnover of microtubules. This study demonstrates that tubulin-GFP fluorescence recovery after photobleaching can be used to assay microtubule dynamics directly in living synapses.

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