F-actin is concentrated in nonrelease domains at frog neuromuscular junctions - PubMed (original) (raw)

F-actin is concentrated in nonrelease domains at frog neuromuscular junctions

A Dunaevsky et al. J Neurosci. 2000.

Abstract

To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with beta-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal.

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Figures

Fig. 1.

Fig. 1.

Longitudinal view through a region of a frog neuromuscular junction. The nerve terminal, capped by a terminal Schwann cell, is separated from the muscle membrane by the synaptic basal lamina. Several release sites in the nerve terminal are filled with synaptic vesicles and are aligned with junctional folds in the muscle membrane. Processes of the terminal Schwann cell encircle the nerve terminal in some nonrelease domains.

Fig. 2.

Fig. 2.

A target-deprived synaptic site is stained by phalloidin in a ladder-like pattern. A, B, Two confocal_z-_sections of a target-deprived nerve terminal stained with rhodamine-conjugated phalloidin to mark F-actin. C, A projection of a _z-_section series of 42 images. A terminal Schwann cell nucleus is marked with an arrow. Scale bar, 10 μm.

Fig. 3.

Fig. 3.

The majority of F-actin stain is external to synaptic vesicle clusters. A target-deprived synaptic site is stained with SV2 (A) to mark synaptic vesicle clusters and with phalloidin (B) to mark F-actin.C, The merged pseudocolor images reveal that the bands of actin are located between synaptic vesicle clusters rather than at the release sites. Scale bar, 5 μm.

Fig. 4.

Fig. 4.

β-Fodrin stain is distributed in a ladder-like pattern at target-deprived synaptic sites and colocalizes with F-actin stain. A–C, Left, Three confocal_z-sections through target-deprived synaptic sites are stained with an antibody directed against β-fodrin. An_arrow marks a nucleus of a terminal Schwann cell. Scale bar, 10 μm. Right, Pseudocolor images of a target-deprived synaptic site stained with an antibody directed against β-fodrin (D) and phalloidin (E). F, A merged image of_D_ and E. Scale bar, 5 μm.

Fig. 5.

Fig. 5.

Bands of F-actin and β-fodrin stain do not persist after denervation of target-deprived synaptic sites. Innervated (A) or 2 week denervated (B, C) synaptic sites are marked with peanut agglutinin (PNA) and stained for actin microfilaments or β-fodrin. Scale bar, 5 μm.

Fig. 6.

Fig. 6.

Terminal Schwann cell stain is not colocalized with bands of F-actin stain. Target-deprived synaptic sites were double-labeled with markers of terminal Schwann cells, either SC-1 or 2A12, and phalloidin. The terminal Schwann cell stain was rarely banded and did not overlap the bands of phalloidin stain. Scale bar, 5 μm.

Fig. 7.

Fig. 7.

The distribution of F-actin at a synaptic site is coextensive with that of synaptic vesicles. Each_z-series of confocal images was resampled along a specified line to yield a longitudinal view of a synaptic site. The view from top to bottom(arrow) shows a section through the nerve terminal toward the presynaptic membrane and synaptic cleft. Each_column of three images (A–C) shows reconstructed line scans of a synaptic site stained both with an antibody to either SV2 or β-fodrin (in green) and markers of either acetylcholine receptors (BTX) or F-actin (in red). The _bottom panels_represent overlays of images from the pairs of stain. Markers of synaptic structures found in distinct locations (A) were resolved in the _z_-axis by this method. F-actin is coextensive with a synaptic vesicle marker (C) and overlaps completely with β-fodrin (B); co-ordinates: _x_-axis, 3 μm;_z_-axis, 0.5 μm.

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