Axon terminals and muscle fibers secrete filamentous proteins into the synaptic cleft at neuromuscular junctions in skeletal muscles. There, the secreted proteins interact with each other and with non-secreted fila- mentous proteins that extend from the plasma membranes of the axon terminal and muscle fiber, altogether forming a stable, tightly adherent network of macromolecules called the synaptic basal lamina. The basal lami- na macromolecules not only serve to hold the membranes of the axon terminal and muscle fiber in close prox- imity for reliable acetylcholine-mediated synaptic impulse transmission, but they also include proteins active in regulating processes involved in synaptic transmission. Agrin secreted by the axon terminal is one such pro- tein. Its C-terminus interacts with the co-receptor proteins Lrp4 (low density lipoprotein receptor-related protein 4) and MuSK (muscle specific kinase), which extend from the muscle fiber?s postsynaptic membrane. This in- teraction directs the formation and maintenance of the postsynaptic apparatus of muscle fibers, which includes aggregates of the acetylcholine receptors in the muscle fibers? plasma membrane. Genetic defects in or auto- immune action against agrin, Lrp4 or MuSK correlate with myasthenias in some humans having the disease. There is evidence that the macromolecular architecture of synaptic basal lamina across vertebrates is highly ordered. However, the architecture has not been characterized by microscopic analysis for the neuromuscular junctions of any species, and, thus, the layout of the basal lamina proteins and their sites of interaction with other proteins within the architecture has not been determined. Much has been learned about synaptic basal lamina proteins, in general, and agrin?s mechanism of action, in particular, from studies on neuromuscular junc- tions of mice. Here, we propose to begin generating a quantitatively accurate 3-D map of the macromolecular architecture of the synaptic basal lamina at neuromuscular junctions of mature mice and to identify those mac- romolecules that contain the C-terminus of neural agrin. For these studies, we will use electron tomography on individual tissue sections from fixed, stained and plastic embedded muscles of normal mice and of mice in which we will genetically engineer the C-terminus of neural agrin to include miniSOG (mini singlet oxygen gen- erator) for histochemical localization. Electron tomography on tissue sections provides the best spatial resolu- tion currently obtainable for in situ 3-D structural analysis. The insertion of miniSOG into filamentous proteins by genetic engineering offers the most reliable method for histochemically localizing specific sites along them. It is expected that this study will provide the basis for us, and others, to map the architecture of the entire syn- aptic basal lamina and establish the complete layout and sites of interaction of neural agrin, Lrp4/MuSK and the other filamentous proteins that constitute the synaptic basal lamina. Such knowledge is essential for a comprehensive understanding of how the neuromuscular junction forms and functions and how its function is affected by disease.
The synaptic cleft at the neuromuscular junction of skeletal muscles is occupied by many filamentous proteins that stably interact to form network of macromolecules, the synaptic basal lamina. The research in this proposal, which will be done on mouse neuromuscular junctions, is aimed at mapping for the first time in any species the macromolecular architecture of the synaptic basal lamina, focusing on regions that contain neural agrin and identifying by miniSOG histochemistry those macromolecules that contain agrin?s C-terminus; agrin?s C-terminus regulates the formation and maintenance of the postsynaptic apparatus in muscle fibers, which is essential for neuromuscular impulse transmission. It is expected that this study will provide the basis for us, and others, to map the macromolecular architecture of the entire synaptic basal lamina and establish the complete layout and sites of interaction of neural agrin, its receptors and the other filamentous proteins that constitute the synaptic basal lamina. Such knowledge is essential for a comprehensive understanding of how the neuromuscular junction forms and functions and how its function is affected by disease such as myasthenias.
| Jung, Jae H; Szule, Joseph A; Stouder, Kylee et al. (2018) Active Zone Material-Directed Orientation, Docking, and Fusion of Dense Core Vesicles Alongside Synaptic Vesicles at Neuromuscular Junctions. Front Neuroanat 12:72 |
