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Active Zone Material-Directed Orientation, Docking, and Fusion o f Dense Core Vesicles Alongside Synaptic Vesicles at Neuromuscular Junctions FRONTIERS IN NEUROANATOMY Jung, J. H., Szule, J. A., Stouder, K., Marshall, R. M., McMahan, U. J. 2018; 12
View details for DOI 10.3389/fnana.2018.00072

View details for Web of Science ID 000444444300001
Variable priming of a docked synaptic vesicle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jung, J. H., Szule, J. A., Marshall, R. M., McMahan, U. J. 2016; 113 (8): E1098-E1107
Abstract

The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM-PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM-PM contact area is dynamic and in equilibrium. The extent of VM-PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca(2+) channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.

View details for DOI 10.1073/pnas.1523054113

View details for Web of Science ID 000370620300021

View details for PubMedID 26858418

View details for PubMedCentralID PMC4776491
Alignment of synaptic vesicle macromolecules with the macromolecules in active zone material that direct vesicle docking. PloS one Harlow, M. L., Szule, J. A., Xu, J., Jung, J. H., Marshall, R. M., McMahan, U. J. 2013; 8 (7)
Abstract

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron's axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ?10% of a vesicle's luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ?25 sites. The assembly's chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly's shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.

View details for DOI 10.1371/journal.pone.0069410

View details for PubMedID 23894473

View details for PubMedCentralID PMC3718691
Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking. PloS one Harlow, M. L., Szule, J. A., Xu, J., Jung, J. H., Marshall, R. M., McMahan, U. J. 2013; 8 (7)
View details for DOI 10.1371/journal.pone.0069410

View details for PubMedID 23894473
Regulation of Synaptic Vesicle Docking by Different Classes of Macromolecules in Active Zone Material PLOS ONE Szule, J. A., Harlow, M. L., Jung, J. H., De-Miguel, F. F., Marshall, R. M., McMahan, U. J. 2012; 7 (3)
Abstract

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.

View details for DOI 10.1371/journal.pone.0033333

View details for Web of Science ID 000303309100034

View details for PubMedID 22438915

View details for PubMedCentralID PMC3306385
Macromolecular Connections of Active Zone Material to Docked Synaptic Vesicles and Presynaptic Membrane at Neuromuscular Junctions of Mouse JOURNAL OF COMPARATIVE NEUROLOGY Nagwaney, S., Harlow, M. L., Jung, J. H., Szule, J. A., Ress, D., Xu, J., Marshall, R. M., McMahan, U. J. 2009; 513 (5): 457-468
Abstract

Electron tomography was used to view macromolecules composing active zone material (AZM) in axon terminals at mouse neuromuscular junctions. Connections of the macromolecules to each other, to calcium channels in the presynaptic membrane, and to synaptic vesicles docked on the membrane prior to fusing with it during synaptic transmission were similar to those of AZM macromolecules at frog neuromuscular junctions previously examined by electron tomography and support the hypothesis that AZM regulates vesicle docking and fusion. A species difference in the arrangement of AZM relative to docked vesicles may help account for a greater vesicle-presynaptic membrane contact area during docking and a greater probability of fusion during synaptic transmission in mouse. Certain AZM macromolecules in mouse were connected to synaptic vesicles contacting the presynaptic membrane at sites where fusion does not occur. These secondary docked vesicles had a different relationship to the membrane and AZM macromolecules than primary docked vesicles, consistent with their having a different AZM-regulated behavior.

View details for DOI 10.1002/cne.21975

View details for Web of Science ID 000263981100002

View details for PubMedID 19226520
Methods for generation high-resolution structural models from electron microscope tomography data. Structure D.B. Ress, M.L. Harlow, R.M. Marshall, U.J. McMahan 2004; 12 (10): 1763-1774
Optimization method for isodensity surface models obtained with electron microscope tomography data 25th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Ress, D., Harlow, M. L., Marshall, R. M., McMahan, U. J. IEEE. 2003: 774?777
View details for Web of Science ID 000189395300205
Macromolecular architecture of presynaptic apparatus as revealed by electron microscope tomography McMahan, U. J. BIOPHYSICAL SOCIETY. 2002: 339A
View details for Web of Science ID 000173252701663
The architecture of active zone material at the frog's neuromuscular junction NATURE Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M., McMahan, U. J. 2001; 409 (6819): 479-484
Abstract

Active zone material at the nervous system's synapses is situated next to synaptic vesicles that are docked at the presynaptic plasma membrane, and calcium channels that are anchored in the membrane. Here we use electron microscope tomography to show the arrangement and associations of structural components of this compact organelle at a model synapse, the frog's neuromuscular junction. Our findings indicate that the active zone material helps to dock the vesicles and anchor the channels, and that its architecture provides both a particular spatial relationship and a structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter by the synaptic vesicles during synaptic transmission.

View details for Web of Science ID 000166570500038

View details for PubMedID 11206537
Regulation of the size and distribution of agrin-induced postsynaptic-like apparatus in adult skeletal muscle by electrical muscle activity MOLECULAR AND CELLULAR NEUROSCIENCE Mathiesen, I., Rimer, M., Ashtari, O., Cohen, I., McMahan, U. J., Lomo, T. 1999; 13 (3): 207-217
Abstract

We compared actylcholine receptor (AChR) aggregates induced by neural agrin released from transfected muscle fibers with AChR aggregates induced by transplanted axons in extrajunctional regions of denervated rat soleus muscles. Both neural agrin and transplanted axons induced multiple, irregularly distributed AChR aggregates on muscle fibers. Direct electrical muscle stimulation of transfected muscles for up to 10 weeks removed all agrin-induced AChR aggregates (the losers) except one (the winner) on many fibers. Axon-induced AChR aggregates underwent comparable selection of winners and losers. The results suggest that agrin and acetylcholine-driven muscle activity provided by transplanted axons are sufficient to elicit in a denervated adult muscle fiber processes that regulate the size and distribution of ectopic neuromuscular junctions.

View details for Web of Science ID 000080443100004

View details for PubMedID 10328882
Automatic acquisition of fiducial markers and alignment of images in tilt series for electron tomography JOURNAL OF ELECTRON MICROSCOPY Ress, D., Harlow, M. L., Schwarz, M., Marshall, R. M., McMahan, U. J. 1999; 48 (3): 277-287
Abstract

Three-dimensional reconstruction of a section of biological tissue by electron tomography requires precise alignment of a series of two-dimensional images of the section made at numerous successive tilt angles. Gold beads on or in the section serve as fiducial markers. A scheme is described that automatically detects the position of these markers and indexes them from image to image. The resulting set of position vectors are arranged in a matrix representation of the tilt geometry and, by inversion, alignment information is obtained. The scheme is convenient, requires little operator time and provides an accuracy of < 2 pixels RMS. A tilt series of 60-70 images can be aligned in approximately 30 min on any modern desktop computer.

View details for Web of Science ID 000080812400012

View details for PubMedID 10425746
Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle MOLECULAR AND CELLULAR NEUROSCIENCE Rimer, M., Cohen, I., Lomo, T., Burden, S. J., McMahan, U. J. 1998; 12 (1-2): 1-15
Abstract

We demonstrate by immunohistochemistry that at least two isoforms of neuregulin (NRG) are concentrated at neuromuscular junctions in adult rat muscles. One is NRGbeta3, a secreted protein which is bound to basal lamina that occupies the synaptic cleft. The other(s), NRG-a, is in the muscle fibers' plasma membrane. We show further that muscle NRG, including NRG-a, is concentrated at postsynaptic-like apparatus induced to form in the extrajunctional region of the soleus muscle by exposure to neural agrin. The agrin-induced postsynaptic-like apparatus also includes aggregates of the NRG receptors erbB2 and erbB3 as does postsynaptic apparatus at neuromuscular junctions. These findings together with those of others suggest a mechanism by which neural agrin induces the expression of epsilon-AChR subunits in postsynaptic-like apparatus, and they support the hypothesis that agrin has a similar function at neuromuscular junctions.

View details for Web of Science ID 000076764900001

View details for PubMedID 9770336
Dissection of active zones at the neuromuscular junction by EM tomography JOURNAL OF PHYSIOLOGY-PARIS Harlow, M., Ress, D., Koster, A., Marshall, R. M., Schwarz, M., McMahan, U. J. 1998; 92 (2): 75-78
Abstract

We used EM tomography to examine the fine structure of the apparently amorphous electron dense material that is seen at active zones of axon terminals when viewed by conventional 2D electron microscopy. Serial 1-nm optical slices from 3D reconstructions of individual thin tissue sections reveal that the material is composed of an interconnecting network of elongate components directly linked to synaptic vesicles and the presynaptic membrane. Each vesicle at the active zone that lies adjacent to the presynaptic plasma membrane has several such connections. Information provided by reconstruction data may be useful in generating experiments aimed at understanding the mechanisms involved in the docking of synaptic vesicles and their exocytosis during synaptic transmission.

View details for Web of Science ID 000076191700004

View details for PubMedID 9782447
Ligands for ErbB-family receptors encoded by a neuregulin-like gene NATURE Chang, H., Riese, D. J., Gilbert, W., Stern, D. F., McMahan, U. J. 1997; 387 (6632): 509-512
Abstract

Neuregulins (also called ARIA, GGF, heregulin or NDF) are a group of polypeptide factors that arise from alternative RNA splicing of a single gene. Through their interaction with the ErbB family of receptors (ErbB2, ErbB3 and ErbB4), neuregulins help to regulate cell growth and differentiation in many tissues. Here we report the cloning of a second neuregulin-like gene, neuregulin-2. The encoded product of the neuregulin-2 gene has a motif structure similar to that of neuregulins and an alternative splicing site in the epidermal growth factor(EGF)-like domain gives rise to two isoforms (alpha and beta). Northern blot and in situ hybridization analysis of adult rat tissues indicate that expression of neuregulin-2 is highest in the cerebellum, and the expression pattern is different from that of neuregulins. Recombinant neuregulin-2beta induces the tyrosine-phosphorylation of ErbB2, ErbB3 and ErbB4 in cell lines expressing all of these ErbB-family receptors. However, in cell lines with defined combinations of ErbBs, neuregulin-2beta only activates those with ErbB3 and/or ErbB4, suggesting that signalling by neuregulin-2 is mediated by ErbB3 and/or ErbB4 receptors.

View details for PubMedID 9168114
GLOBULAR AND ASYMMETRIC ACETYLCHOLINESTERASE IN THE SYNAPTIC BASAL LAMINA OF SKELETAL-MUSCLE JOURNAL OF CELL BIOLOGY Anglister, L., Haesaert, B., McMahan, U. J. 1994; 125 (1): 183-196
Abstract

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction-specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.

View details for Web of Science ID A1994NE03600015

View details for PubMedID 8138570

View details for PubMedCentralID PMC2120017
ISOLATION AND CHARACTERIZATION OF A CDNA THAT ENCODES AN AGRIN HOMOLOG IN THE MARINE RAY MOLECULAR AND CELLULAR NEUROSCIENCE Smith, M. A., MAGILLSOLC, C., Rupp, F., Yao, Y. M., SCHILLING, J. W., Snow, P., McMahan, U. J. 1992; 3 (5): 406-417
Abstract

Agrin, the protein thought to trigger motor neuron-induced aggregation of postsynaptic molecules at the developing neuromuscular junction, has been purified from the synapse-rich electric organ of the marine ray. In order to study agrin's role in synaptogenesis and to examine its relationship to antigenically similar proteins, we isolated from a marine ray library a partial cDNA, OL4, which codes for a member of the agrin protein family. Sequence analysis shows that agrin and agrin-related proteins contain regions similar to basal lamina proteins and other secreted molecules including laminin, epidermal growth factor, and pancreatic secretory trypsin inhibitors. Northern blot analysis revealed transcripts in several different tissues, but the highest levels of expression are in brain and spinal cord. In situ hybridization studies demonstrate that agrin/agrin-related mRNAs are present in motor neurons that innervate the electric organ and skeletal muscle. They also reveal that agrin/agrin-related transcripts have a broad distribution in neurons and nonneural cells in the CNS, raising the possibility that agrin and/or agrin-related proteins mediate formation of the postsynaptic apparatus at neuron-to-neuron synapses.

View details for Web of Science ID A1992JP07300004

View details for PubMedID 19912884
Agrin isoforms and their role in synaptogenesis. Current opinion in cell biology McMahan, U. J., HORTON, S. E., Werle, M. J., Honig, L. S., Kr�ger, S., Ruegg, M. A., Escher, G. 1992; 4 (5): 869-874
Abstract

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.

View details for PubMedID 1329871
AGRIN RELEASED BY MOTOR NEURONS INDUCES THE AGGREGATION OF ACETYLCHOLINE-RECEPTORS AT NEUROMUSCULAR-JUNCTIONS NEURON Reist, N. E., Werle, M. J., McMahan, U. J. 1992; 8 (5): 865-868
Abstract

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.

View details for Web of Science ID A1992HV46700005

View details for PubMedID 1316763
THE AGRIN GENE CODES FOR A FAMILY OF BASAL LAMINA PROTEINS THAT DIFFER IN FUNCTION AND DISTRIBUTION NEURON Ruegg, M. A., Tsim, K. W., HORTON, S. E., Kroger, S., Escher, G., GENSCH, E. M., McMahan, U. J. 1992; 8 (4): 691-699
Abstract

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

View details for Web of Science ID A1992HQ18800008

View details for PubMedID 1314621
CDNA THAT ENCODES ACTIVE AGRIN NEURON Tsim, K. W., Ruegg, M. A., Escher, G., Kroger, S., McMahan, U. J. 1992; 8 (4): 677-689
Abstract

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

View details for Web of Science ID A1992HQ18800007

View details for PubMedID 1314620
MOLECULES THAT INDUCE THE FORMATION OF SYNAPTIC APPARATUS MEETING ON PLASTICITY OF MOTONEURONAL CONNECTIONS Werle, M. J., McMahan, U. J. ELSEVIER SCIENCE PUBL B V. 1991: 269?273
View details for Web of Science ID A1991BV30S00029
SYNTHESIS AND TRANSPORT OF AGRIN-LIKE MOLECULES IN MOTOR NEURONS DISCUSSION MEETING ON SYNAPSE FORMATION MAGILLSOLC, C., McMahan, U. J. COMPANY OF BIOLOGISTS LTD. 1990: 1?10
Abstract

Several lines of evidence indicate that agrin, or a protein very similar to it, directs the formation and maintenance of the postsynaptic apparatus at the neuromuscular junction. We discuss the results of studies involving immunohistochemical, biochemical and in situ hybridization techniques that support the hypothesis that agrin or agrin-like molecules active at the junction are produced by motor neurons.

View details for Web of Science ID A1990EG83700002

View details for PubMedID 2177765
AGRIN-LIKE MOLECULES IN MOTOR NEURONS 13TH GIF CONF ON NEUROBIOLOGY : EXPRESSION AND ASSEMBLY OF A FUNCTIONAL NEURON MAGILLSOLC, C., McMahan, U. J. MASSON EDITEUR. 1990: 78?81
Abstract

According to the agrin hypothesis molecules that mediate the nerve-induced aggregation of acetylcholine receptors and acetylcholinesterase on developing and regenerating skeletal muscle fibers are similar or identical to agrin, a protein extracted from the electric organ of marine rays. Here we present evidence that agrin is highly concentrated in the cell bodies of motor neurons and is transported to axon terminals which is consistent with the agrin hypothesis.

View details for Web of Science ID A1990DG73700009

View details for PubMedID 2193148
MOLECULES IN BASAL LAMINA THAT DIRECT THE FORMATION OF SYNAPTIC SPECIALIZATIONS AT NEUROMUSCULAR-JUNCTIONS DEVELOPMENTAL NEUROSCIENCE McMahan, U. J., Wallace, B. G. 1989; 11 (4-5): 227-247
View details for Web of Science ID A1989AH57000002

View details for PubMedID 2676454
MOTOR NEURONS CONTAIN AGRIN-LIKE MOLECULES JOURNAL OF CELL BIOLOGY MAGILLSOLC, C., McMahan, U. J. 1988; 107 (5): 1825-1833
Abstract

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.

View details for Web of Science ID A1988Q766200022

View details for PubMedID 2846587
IDENTIFICATION OF AGRIN, A SYNAPTIC ORGANIZING PROTEIN FROM TORPEDO ELECTRIC ORGAN JOURNAL OF CELL BIOLOGY Nitkin, R. M., Smith, M. A., Magill, C., Fallon, J. R., Yao, Y. M., Wallace, B. G., McMahan, U. J. 1987; 105 (6): 2471-2478
Abstract

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."

View details for Web of Science ID A1987L414400004

View details for PubMedID 2826489
AGRIN-LIKE MOLECULES AT SYNAPTIC SITES IN NORMAL, DENERVATED, AND DAMAGED SKELETAL-MUSCLES JOURNAL OF CELL BIOLOGY Reist, N. E., Magill, C., McMahan, U. J. 1987; 105 (6): 2457-2469
Abstract

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.

View details for Web of Science ID A1987L414400003

View details for PubMedID 2826488
IDENTIFICATION OF AGRIN IN ELECTRIC ORGAN EXTRACTS AND LOCALIZATION OF AGRIN-LIKE MOLECULES IN MUSCLE AND CENTRAL-NERVOUS-SYSTEM JOURNAL OF EXPERIMENTAL BIOLOGY Smith, M. A., Yao, Y. M., Reist, N. E., Magill, C., Wallace, B. G., McMahan, U. J. 1987; 132: 223-230
Abstract

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.

View details for Web of Science ID A1987K564500016

View details for PubMedID 2828510
Agrin PROGRESS IN BRAIN RESEARCH <D> Magill, C., Reist, N. E., Fallon, J. R., Nitkin, R. M., Wallace, B. G., McMahan, U. J. 1987; 71: 391-396
View details for Web of Science ID 000207653600033

View details for PubMedID 3035610
CELL ACCUMULATION IN THE JUNCTIONAL REGION OF DENERVATED MUSCLE JOURNAL OF CELL BIOLOGY Connor, E. A., McMahan, U. J. 1987; 104 (1): 109-120
Abstract

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.

View details for Web of Science ID A1987F629900012

View details for PubMedID 3491825

View details for PubMedCentralID PMC2117033
AGGREGATES OF ACETYLCHOLINESTERASE INDUCED BY ACETYLCHOLINE RECEPTOR AGGREGATING FACTOR NATURE Wallace, B. G., Nitkin, R. M., Reist, N. E., Fallon, J. R., Moayeri, N. N., McMahan, U. J. 1985; 315 (6020): 574-577
Abstract

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.

View details for Web of Science ID A1985AKB6600041

View details for PubMedID 4010772
BASAL LAMINA DIRECTS ACETYLCHOLINESTERASE ACCUMULATION AT SYNAPTIC SITES IN REGENERATING MUSCLE JOURNAL OF CELL BIOLOGY Anglister, L., McMahan, U. J., Marshall, R. M. 1985; 101 (3): 735-743
Abstract

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.

View details for Web of Science ID A1985APU0400006

View details for PubMedID 3875617

View details for PubMedCentralID PMC2113729
ACETYLCHOLINE RECEPTOR AGGREGATING FACTOR IS SIMILAR TO MOLECULES CONCENTRATED AT NEUROMUSCULAR-JUNCTIONS NATURE Fallon, J. R., Nitkin, R. M., Reist, N. E., Wallace, B. G., McMahan, U. J. 1985; 315 (6020): 571-574
Abstract

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.

View details for Web of Science ID A1985AKB6600040

View details for PubMedID 3892302
THE INFLUENCE OF BASAL LAMINA ON THE ACCUMULATION OF ACETYLCHOLINE-RECEPTORS AT SYNAPTIC SITES IN REGENERATING MUSCLE JOURNAL OF CELL BIOLOGY McMahan, U. J., Slater, C. R. 1984; 98 (4): 1453-1473
Abstract

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.

View details for Web of Science ID A1984SM02900032

View details for PubMedID 6609164

View details for PubMedCentralID PMC2113239
EXTRACELLULAR-MATRIX COMPONENTS INVOLVED IN NEUROMUSCULAR-TRANSMISSION AND REGENERATION CIBA FOUNDATION SYMPOSIA Anglister, L., McMahan, U. J. 1984; 108: 163-178
Abstract

The portion of a skeletal muscle fibre's basal lamina sheath that lies in the synaptic cleft at the neuromuscular junction contains a high concentration of certain molecules that distinguish it from non-junctional portions of the sheath. Among the molecules are acetylcholinesterase, which terminates the action of the transmitter, acetylcholine, on the postsynaptic membrane, and factors that direct differentiation at neuromuscular junctions regenerating after trauma. In this communication the evidence that acetylcholinesterase and synapse differentiation factors are associated with synaptic cleft basal lamina is reviewed and the results of current experiments aimed at characterizing these extracellular matrix molecules are described.

View details for Web of Science ID A1984TY48700010

View details for PubMedID 6097420
COMPONENTS OF TORPEDO ELECTRIC ORGAN AND MUSCLE THAT CAUSE AGGREGATION OF ACETYLCHOLINE-RECEPTORS ON CULTURED MUSCLE-CELLS JOURNAL OF CELL BIOLOGY Godfrey, E. W., Nitkin, R. M., Wallace, B. G., Rubin, L. L., McMahan, U. J. 1984; 99 (2): 615-627
Abstract

The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.

View details for Web of Science ID A1984TE96000027

View details for PubMedID 6746740
MOLECULAR-COMPONENTS OF THE SYNAPTIC BASAL LAMINA THAT DIRECT DIFFERENTIATION OF REGENERATING NEUROMUSCULAR-JUNCTIONS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Nitkin, R. M., Wallace, B. G., Spira, M. E., Godfrey, E. W., McMahan, U. J. 1983; 48: 653-665
Abstract

Results of experiments outlined here provide evidence that components of the myofiber basal lamina sheath direct the formation of active zones in regenerating motor nerve terminals and the development of infoldings and the aggregation of AChRs in the plasma membrane of regenerating myofibers. As a step toward identifying the basal lamina molecules that aggregate AChRs, we are now studying an ECM fraction from the Torpedo electric organ that causes AChRs to aggregate on cultured myotubes. We have solubilized and purified the electric organ AChR-aggregating molecules over 1000-fold. Only nanogram amounts of the most purified extracts are required to cause detectable AChR aggregation. We have also shown that similar activity can be extracted in relatively small amounts from muscle. Antiserum raised against the partially purified electric organ material completely blocked and immunoprecipitated the AChR-aggregating activity in extracts of the electric organ and muscle and bound to components of the basal lamina of frog muscle fibers. Although several polypeptides are present in our most purified extracts, an antiserum against polypeptides in the range of 80 kD completely blocked AChR aggregation by soluble extracts of the electric organ. These findings demonstrate the feasibility of isolating molecules from the synapse-rich electric organ that cause AChR aggregation and comparing them by immunological techniques with those in basal lamina at the neuromuscular junction.

View details for Web of Science ID A1983SX49600068

View details for PubMedID 6586382
FACTORS THAT INFLUENCE REGENERATION OF THE NEUROMUSCULAR-JUNCTION JOURNAL OF EXPERIMENTAL BIOLOGY McMahan, U. J., EDGINGTON, D. R., Kuffler, D. P. 1980; 89 (DEC): 31-?
Abstract

Regeneration of neuromuscular junctions after trauma occurs in an orderly way and relies on communication between nerve and muscle. This paper summarizes evidence that factors which direct the growth and differentiation of both pre- and postsynaptic components of regenerating neuromuscular junctions are associated with the extracellular matrix of muscles.

View details for Web of Science ID A1980LB27200005

View details for PubMedID 7009777
ACETYLCHOLINE RECEPTORS IN REGENERATING MUSCLE ACCUMULATE AT ORIGINAL SYNAPTIC SITES IN THE ABSENCE OF THE NERVE JOURNAL OF CELL BIOLOGY Burden, S. J., Sargent, P. B., McMahan, U. J. 1979; 82 (2): 412-425
Abstract

We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.

View details for Web of Science ID A1979HE13500009

View details for PubMedID 479308

Uel Jackson McMahan

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