Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

Hiroshi Nishimune,Yomna Badawi,Kazuhiro Shigemoto,Shuuichi Mori

doi:10.1038/srep27935

Hiroshi Nishimune, Yomna Badawi + Show 2 more

Open Access

https://doi.org/10.1038/srep27935

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Abstract

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.

Highlights

We previously described the distribution pattern of the active zone proteins Bassoon, Piccolo, and P/Q-type voltage-gated calcium channels (VGCCs) in mouse NMJs as numerous small puncta distributing as discrete protein accumulations[28,29,30]
In adult NMJs, P/Q-type VGCCs were distributed in a punctate pattern that aligned with bright lines of α-bungarotoxin staining pattern as detected via confocal microscopy (Fig. 1)
The alignment of VGCC puncta with junctional folds is consistent with the alignment of presynaptic active zones with junctional folds observed using electron microscopy[52] and electron tomography[9] because these calcium channels are thought to be in or near the active zones

Summary

Introduction

We previously described the distribution pattern of the active zone proteins Bassoon, Piccolo, and P/Q-type VGCCs in mouse NMJs as numerous small puncta distributing as discrete protein accumulations[28,29,30]. The active zone density decreases in aged mice and aged rats compared to the active zone density in adult NMJs29,30. These analyses were limited by the diffraction-limited resolution of confocal microscopy. We identified an unexpected non-overlapping side-by-side distribution pattern of these proteins in mouse NMJs

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