Dynamin inhibition blocks the formation of bulk endosomes and promotes intraterminal tubulation.

<p>NMJ preparations were treated with 30 µM dyngo-4a for 40 min in Ringer's solution (2 mM Ca<sup>2+)</sup> followed by stimulation at 20 Hz for 10 min. A pulse of FM1-43 was applied for the last 5 min of stimulation, followed by washing with cold Ca<sup>2+</sup>-free Ringer's solution. Fresh Ringer's solution containing 30 µM dyngo-4a was then reapplied to the preparation, followed by confocal imaging. (<b>A</b>) Representative image of control unstimulated nerve terminal showing membrane localization of FM1-43. (<b>B</b>) Stimulation of untreated nerve terminals at low frequency (1 Hz) revealed the appearance of classical FM1-43-positive bands representative of synaptic vesicle clusters. (<b>C</b>) Stimulation of nerve terminals at 20 Hz revealed the appearance of large FM1-43-positive donut-shaped intraterminal structures. (<b>D</b>) Unstimulated dyngo-4a-treated nerve terminals exhibited similar plasma membrane FM1-43 staining to untreated terminals. Dyngo-4a-treated nerve terminals stimulated at 20 Hz exhibited a striking network of FM1-43-positive tubules that appeared to be connected to the plasma membrane (<b>E–I</b>). Filament trace modeling (<b>F</b> and <b>G</b>) demonstrates the reticulated nature of the branched tubular networks induced by dyngo-4a at the amphibian NMJ. (<b>H</b> and <b>I</b>), Depth coding analysis of nerve terminals revealed that the tubular networks were mostly located intraterminally, with most tubules residing within the green-yellow region (0.5 µm away from the plasma membrane). In some instances, the termination points of individual branches were located within the red region, indicating membrane localization (white arrows) of these branch terminals. Scale bars 5 µm.</p>