Abstract
Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.
Highlights
The synapses formed between motoneurons and muscle fibers, termed neuromuscular junctions (NMJs) are responsible for the transmission of signals from the nerve to the muscle, enabling crucial functions of the organism, such as movement or respiration [1,2]
We show that drebrin downregulation or inhibition of its interaction with actin impairs the targeting of microtubules to acetylcholine receptors (AChRs) clusters and disrupts microtubule organization at the postsynaptic machinery in vitro
To test whether drebrin will be recruited to different structural domains during NMJ remodeling, we explored the localization of this protein in triangularis sterni muscle at P9, P21, and P45 with a special emphasis on the postsynaptic machinery, labeled with fluorescent bungarotoxin (BTX) that binds to AChRs
Summary
The synapses formed between motoneurons and muscle fibers, termed neuromuscular junctions (NMJs) are responsible for the transmission of signals from the nerve to the muscle, enabling crucial functions of the organism, such as movement or respiration [1,2]. NMJs undergo intensive remodeling starting around P7 up to adulthood (around P30) and during that process, they transform from simple oval plaques into ‘pretzel-like’ structures. Structural transformation of the NMJ into an elaborate, perforated AChR array mirroring the shape of the nerve terminal is pivotal for efficient synaptic transmission [10]. It is postulated that dynamic actin-rich structures, termed synaptic podosomes, remodel the maturing postsynaptic machinery in cultured myotubes and contribute to the formation of the perforations between AChRs [17,18,19]. The potential role of synaptic podosomes at NMJs still requires verification
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