In vivo assay of presynaptic microtubule cytoskeleton dynamics in Drosophila

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 posttranslationally 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.

Kismet Positively Regulates Glutamate Receptor Localization and Synaptic Transmission at the Drosophila Neuromuscular Junction

“…Both the actin and microtubule cytoskeletons are important for the structure, stability, and the maintenance of synapses (for review see [45] ). The microtubule cytoskeleton is essential for both synaptic function and muscle morphology as it forms and provides stability to structural components of neurons and synapses [46] , [47] . To determine whether kis influences the dynamics of the synaptic cytoskeleton like other chromatin remodeling enzymes [48] – [50] , we labeled both the actin and microtubule cytoskeletons at the NMJ using two kis mutant alleles.…”

Formin-Dependent Synaptic Growth: Evidence That Dlar Signals via Diaphanous to Modulate Synaptic Actin and Dynamic Pioneer Microtubules

“…Although a dynamic population of microtubules has been imaged within dendrites and axons of Drosophila neurons (Rolls et al, 2007), this population of microtubules has yet to be characterized in detail within the presynaptic nerve terminal. A recent study has examined the fluorescence recovery after photobleaching for GFP-tagged tubulin and provided evidence for the existence of dynamic presynaptic microtubules (Yan and Broadie, 2007). Here we first set out to image and quantify this dynamic population of presynaptic microtubules at the Drosophila NMJ.…”

Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

“…1i), possibly because microtubules were not sufficiently dynamic to repopulate the area on this timescale. We cannot rule out that despite efforts to minimize SiR-tubulin-induced microtubule stabilization, there is still some stabilizing effect that would be expected to delay recovery 29 .…”