Podosomes are present in a postsynaptic apparatus and participate in its maturation

Abstract

A critical step in synapse formation is the clustering of neurotransmitter receptors in the postsynaptic membrane, directly opposite the nerve terminal. At the neuromuscular junction, a widely studied model synapse, acetylcholine receptors (AChRs) initially aggregate to form an ovoid postsynaptic plaque. As the synapse matures, the plaque becomes perforated and is eventually transformed into a complex, branched structure. We found that this transformation also occurs in myotubes cultured in the absence of neurons, and used this system to seek machinery that orchestrates postsynaptic maturation. We show that perforations in the AChR aggregate bear structures resembling podosomes, dynamic actin-rich adhesive organelles involved in matrix remodeling in non-neuronal cells but not described in neural structures. The location and dynamics of synaptic podosomes are spatiotemporally correlated with changes in AChR aggregate topology, and pharmacological disruption of podosomes leads to rapid alterations in AChR organization. Our results indicate that synaptic podosomes play critical roles in maturation of the postsynaptic membrane.

Fig. 1.

Actin–rich puncta lie within the perforations of AChR aggregates. C2C12 myotubes cultured on laminin were incubated with BTX (red) and phalloidin (green) to label AChR and F-actin, respectively. (A) In immature AChR plaques (day 3 after cell fusion) fine actin cables lie below aggregates. (B) As plaques become perforated (day 4 after cell fusion), actin-rich puncta appear within perforations. (C) As aggregates become branched (day 5 after cell fusion), they bear multiple actin-rich puncta. (D) The number of actin-rich puncta increases during maturation of the AChR clusters in C2C12 myotubes (n = >45 per time point). (E) Similar actin-rich structures were found in perforations at the developing NMJ (postnatal day 8). Tibialis anterior muscle was infected with adenovirus expressing β–actin-GFP (green) and counterstained with BTX (red). Area bracketed in (E) is shown in higher magnification in (F). (Scale bars, 5 μm.)

Fig. 2.

Components of synaptic podosomes. C2C12 myotubes were labeled with BTX (red) and antibodies to the indicated components (green): vinculin (A), dynamin (B), LL5β and actin (C). (Scale bar, 5 μm.) (D) Schematic localization of proteins in synaptic podosomes. AChR-rich region (blue) also contains dystroglycan, α-dystrobrevin, rapsyn, integrin β1, and Src. Perforations in the receptor clusters are occupied by cores (red) containing β-actin, Arp2, NCK, cortactin, myosin IIA, dynamin, Tks5, and Src. The core is surrounded by a cortex (green) rich in talin, vinculin, paxillin, LL5β, integrin β1, Src, and utrophin.

Fig. 3.

Synaptic podosomes are sites of adhesion. Adhesion of C2C12 myotubes to substratum revealed by internal reflection microscopy (IRM). In mature clusters, podosomes are sites of strong adhesion. Dark spots in IRM image reveal close apposition to substratum. (Scale bar, 5 μm.)

Fig. 4.

Synaptic podosomes remodel extracellular matrix. (A and B) C2C12 myotubes were stained for AChR and laminin α5. In immature clusters, laminin α5 (green) and β2 (Fig. S5A) are distributed underneath the AChRs (red) (A). After formation of podosomes, laminin α5 (B) is lost from sites of perforations in the AChR. A thin ring of laminins often remains at the edge of the perforation, corresponding to sites of increased concentrations of integrin β1 (Fig. S3). (C) Exogenous laminin 1 (green) that had been used to coat slide surface before plating cells was also removed from beneath AChR perforations. (Scale bar, 5 μm.)

Fig. 5.

Enhanced endocytosis associated with synaptic podosomes. (A) C2C12 myotubes expressing AP2 sigma subunit. AP2 was concentrated at the AChR clusters (Left; lower magnification; dotted lines outline the location of myotube) and within the clusters, they were enriched around podosomes in the AChRs (Center and Right; higher magnification). (B) Internalization of endocytic tracers, transferrin, occurs at similar locations. (Scale bar, 5 μm.)

Fig. 6.

Dynamic behavior of podosomes revealed by time-lapse imaging. (A) Selected frames from a 2.5-h imaging session. (B) Each podosome was numbered and its presence through the session is plotted. While some podosomes are stable, others are dynamic. Podosomes often disappear then reappear at the same location, as shown for podosome 2 (yellow arrow in A) or 4 (blue arrow in A). (Scale bar, 5 μm.)

Fig. 7.

Appearance of podosomes correlates with maturation of AChR clusters. Time-lapse analysis revealed that podosomes (green arrow) appear underneath AChR clusters at sites where perforations subsequently form (red arrow no. 1). Each perforation formed during analyzed period was marked with red arrow and numbered. While perforation 1 was long lived, other collapsed soon after podosomes disappeared. (Scale bar, 5 μm.)

Fig. 8.

Loss of podosomes leads to altered distribution of AChRs. (A and B) Incubation with Src family kinase inhibitor PP2 led to disassembly of podosomes as shown by loss of actin puncta and dispersal of vinculin. This disruption was associated with redistribution of AChRs into the perforation. (C) Quantification of PP2-depemdent alteration in AChRs distribution. Graph shows measurements from a total of three cultures. (D) Schematic summary of results providing a model for podosomes dependent remodeling of AChR clusters. Muscle cells deposit synaptic basal lamina components underneath AChR plaques (blue). Small actin complexes associated with AChRs give rise to podosomes that grow and form perforations in the cluster (2). Podosomes secrete proteases that remodel basal lamina; along with local endocytosis, this leads to remodeling of the postsynaptic membrane (3). Growth of podosomes leads to formation of branches (4) whereas disassembly of podosomes leads to entry of AChR into perforations (5). With time, however perforations become stable and no longer require podosomes for their maintenance (6). (Scale bar, 5 μm.)