Synaptic nanomodules underlie the organization and plasticity of spine synapses.

Experience results in long-lasting changes in dendritic spine size, yet how the molecular architecture of the synapse responds to plasticity remains poorly understood. Here a combined approach of multicolor stimulated emission depletion microscopy (STED) and confocal imaging in rat and mouse demonstrates that structural plasticity is linked to the addition of unitary synaptic nanomodules to spines. Spine synapses in vivo and in vitro contain discrete and aligned subdiffraction modules of pre- and postsynaptic proteins whose number scales linearly with spine size. Live-cell time-lapse super-resolution imaging reveals that NMDA receptor-dependent increases in spine size are accompanied both by enhanced mobility of pre- and postsynaptic modules that remain aligned with each other and by a coordinated increase in the number of nanomodules. These findings suggest a simplified model for experience-dependent structural plasticity relying on an unexpectedly modular nanomolecular architecture of synaptic proteins

Positive surface charge of GluN1 N-terminus mediates the direct interaction with EphB2 and NMDAR mobility.

Localization of the N-methyl-D-aspartate type glutamate receptor (NMDAR) to dendritic spines is essential for excitatory synaptic transmission and plasticity. Rather than remaining trapped at synaptic sites, NMDA receptors undergo constant cycling into and out of the postsynaptic density. Receptor movement is constrained by protein-protein interactions with both the intracellular and extracellular domains of the NMDAR. The role of extracellular interactions on the mobility of the NMDAR is poorly understood. Here we demonstrate that the positive surface charge of the hinge region of the N-terminal domain in the GluN1 subunit of the NMDAR is required to maintain NMDARs at dendritic spine synapses and mediates the direct extracellular interaction with a negatively charged phospho-tyrosine on the receptor tyrosine kinase EphB2. Loss of the EphB-NMDAR interaction by either mutating GluN1 or knocking down endogenous EphB2 increases NMDAR mobility. These findings begin to define a mechanism for extracellular interactions mediated by charged domains

Nanoscale rules governing the organization of glutamate receptors in spine synapses are subunit specific

Heterotetrameric glutamate receptors are essential for the development, function, and plasticity of spine synapses but how they are organized to achieve this is not known. Here we show that the nanoscale organization of glutamate receptors containing specific subunits define distinct subsynaptic features. Glutamate receptors containing GluA2 or GluN1 subunits establish nanomodular elements precisely positioned relative to Synaptotagmin-1 positive presynaptic release sites that scale with spine size. Glutamate receptors containing GluA1 or GluN2B specify features that exhibit flexibility: GluA1-subunit containing AMPARs are found in larger spines, while GluN2B-subunit containing NMDARs are enriched in the smallest spines with neither following a strict modular organization. Given that the precise positioning of distinct classes of glutamate receptors is linked to diverse events including cell death and synaptic plasticity, this unexpectedly robust synaptic nanoarchitecture provides a resilient system, where nanopositioned glutamate receptor heterotetramers define specific subsynaptic regions of individual spine synapses

Ephrin-B3 controls excitatory synapse density through cell-cell competition for EphBs

Cortical networks are characterized by sparse connectivity, with synapses found at only a subset of axo-dendritic contacts. Yet within these networks, neurons can exhibit high connection probabilities, suggesting that cell-intrinsic factors, not proximity, determine connectivity. Here, we identify ephrin-B3 (eB3) as a factor that determines synapse density by mediating a cell-cell competition that requires ephrin-B-EphB signaling. In a microisland culture system designed to isolate cell-cell competition, we find that eB3 determines winning and losing neurons in a contest for synapses. In a Mosaic Analysis with Double Markers (MADM) genetic mouse model system in vivo the relative levels of eB3 control spine density in layer 5 and 6 neurons. MADM cortical neurons in vitro reveal that eB3 controls synapse density independently of action potential-driven activity. Our findings illustrate a new class of competitive mechanism mediated by trans-synaptic organizing proteins which control the number of synapses neurons receive relative to neighboring neurons

Defects in synapse structure and function precede motor neuron degeneration in Drosophila models of FUS-related ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. We show here that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by \u3e80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS

Neuron Glia-Related Cell Adhesion Molecule (NrCAM) Promotes Topographic Retinocollicular Mapping

NrCAM (Neuron-glial related cell adhesion molecule), a member of the L1 family of cell adhesion molecules, reversibly binds ankyrin and regulates axon growth, but it has not been studied for a role in retinotopic mapping. During development of retino-collicular topography, NrCAM was expressed uniformly in retinal ganglion cells (RGCs) along both mediolateral and anteroposterior retinal axes, and was localized on RGC axons within the optic tract and superior colliculus (SC). Anterograde tracing of RGC axons in NrCAM null mutant mice at P10, when the map resembles its mature form, revealed laterally displaced ectopic termination zones (eTZs) of axons from the temporal retina, indicating defective mediolateral topography, which is governed by ephrinB/EphBs. Axon tracing at P2 revealed that interstitial branch orientation of ventral-temporal RGC axons in NrCAM null mice was compromised in the medial direction, likely accounting for displacement of eTZs. A similar retinocollicular targeting defect in EphB mutant mice suggested that NrCAM and EphB interact to regulate mediolateral retino-collicular targeting. We found that EphB2 tyrosine kinase but not an EphB2 kinase dead mutant, phosphorylated NrCAM at a conserved tyrosine residue in the FIGQY ankyrin binding motif, perturbing ankyrin recruitment in NrCAM transfected HEK293 cells. Furthermore, the phosphorylation of NrCAM at FIGQY in SC was decreased in EphB1/3 and EphB1/2/3 null mice compared to WT, while phospho-FIGQY of NrCAM in SC was increased in EphB2 constitutively active (F620D/F620D) mice. These results demonstrate that NrCAM contributes to mediolateral retinocollicular axon targeting by regulating RGC branch orientation through a likely mechanism in which ephrinB/EphB phosphorylates NrCAM to modulate linkage to the actin cytoskeleton

Neuronal activity moves protein palmitoylation into the synapse

Many neuronal proteins undergo lipid modification that regulates their function and subcellular localization. One such modification is palmitoylation, which is mediated by a large class of protein palmitoyl acyltransferases (PATs). Now, a paper in this issue (Noritake et al. 2009. J. Cell Biol. doi:10.1083/jcb.200903101) demonstrates that the localization of the PAT DHHC2 is regulated by neuronal activity and thereby selectively controls the palmitoylation and subsequent accumulation of specific proteins in the synapse

MAxSIM: Multi-Angle-Crossing Structured Illumination Microscopy With Height-Controlled Mirror for 3D Topological Mapping of Live Cells

Mapping 3D plasma membrane topology in live cells can bring unprecedented insights into cell biology. Widefield-based super-resolution methods such as 3D-structured illumination microscopy (3D-SIM) can achieve twice the axial ( ~ 300 nm) and lateral ( ~ 100 nm) resolution of widefield microscopy in real time in live cells. However, twice-resolution enhancement cannot sufficiently visualize nanoscale fine structures of the plasma membrane. Axial interferometry methods including fluorescence light interference contrast microscopy and its derivatives (e.g., scanning angle interference microscopy) can determine nanoscale axial locations of proteins on and near the plasma membrane. Thus, by combining super-resolution lateral imaging of 2D-SIM with axial interferometry, we developed multi-angle-crossing structured illumination microscopy (MAxSIM) to generate multiple incident angles by fast, optoelectronic creation of diffraction patterns. Axial localization accuracy can be enhanced by placing cells on a bottom glass substrate, locating a custom height-controlled mirror (HCM) at a fixed axial position above the glass substrate, and optimizing the height reconstruction algorithm for noisy experimental data. The HCM also enables imaging of both the apical and basal surfaces of a cell. MAxSIM with HCM offers high-fidelity nanoscale 3D topological mapping of cell plasma membranes with near-real-time ( ~ 0.5 Hz) imaging of live cells and 3D single-molecule tracking

Ephrin-B2 Promotes Nociceptive Plasticity and Hyperalgesic Priming Through EphB2-MNK-eIF4E Signaling in Both Mice and Humans

Ephrin-B-EphB signaling can promote pain through ligand-receptor interactions between peripheral cells, like immune cells expressing ephrin-Bs, and EphB receptors expressed by DRG neurons. Previous studies have shown increased ephrin-B2 expression in peripheral tissues like synovium of rheumatoid and osteoarthritis patients, indicating the clinical significance of this signaling. The primary goal of this study was to understand how ephrin-B2 acts on mouse and human DRG neurons, which express EphB receptors, to promote pain and nociceptor plasticity. We hypothesized that ephrin-B2 would promote nociceptor plasticity and hyperalgesic priming through MNK-eIF4E signaling, a critical mechanism for nociceptive plasticity induced by growth factors, cytokines and nerve injury. Both male and female mice developed dose-dependent mechanical hypersensitivity in response to ephrin-B2, and both sexes showed hyperalgesic priming when challenged with PGE2 injection either to the paw or the cranial dura. Acute nociceptive behaviors and hyperalgesic priming were blocked in mice lacking MNK1 (Mknk1 knockout mice) and by eFT508, a specific MNK inhibitor. Sensory neuron-specific knockout of EphB2 using Pirt-Cre demonstrated that ephrin-B2 actions require this receptor. In Ca2+-imaging experiments on cultured DRG neurons, ephrin-B2 treatment enhanced Ca2+ transients in response to PGE2 and these effects were absent in DRG neurons from MNK1-/- and EphB2-PirtCre mice. In experiments on human DRG neurons, ephrin-B2 increased eIF4E phosphorylation and enhanced Ca2+ responses to PGE2 treatment, both blocked by eFT508. We conclude that ephrin-B2 acts directly on mouse and human sensory neurons to induce nociceptor plasticity via MNK-eIF4E signaling, offering new insight into how ephrin-B signaling promotes pain