Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 10(4)-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

Factors that control differentiation of presynaptic and postsynaptic elements into excitatory or inhibitory synapses are poorly defined. Here we show that the postsynaptic density (PSD) proteins PSD-95 and neuroligin-1 (NLG) are critical for dictating the ratio of excitatory-to-inhibitory synaptic contacts. Exogenous NLG increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory synaptic currents. In contrast, PSD-95 overexpression enhanced excitatory synapse size and miniature frequency, but reduced the number of inhibitory synaptic contacts. Introduction of PSD-95 with NLG augmented synaptic clustering of NLG and abolished NLG effects on inhibitory synapses. Interfering with endogenous PSD-95 expression alone was sufficient to reduce the ratio of excitatory-to-inhibitory synapses. These findings elucidate a mechanism by which the amounts of specific elements critical for synapse formation control the ratio of excitatory-to-inhibitory synaptic input.S ynapse formation involves stabilization of initial sites of contact between axons and dendrites, followed by recruitment of specific protein complexes to newly formed presynaptic and postsynaptic structures (1-4). Neuronal contact formation is spatially and temporally controlled by changes in protein content and shape at areas of contact (5, 6). The total number of synapses formed and ratio of excitatory-to-inhibitory synaptic inputs a neuron receives are factors critical for determining neuronal excitability. Appropriate synthesis and recruitment of specific factors important for building synaptic contacts are thought to power this process. However, the identity of molecules that dictate the balance between excitatory and inhibitory synaptic contacts remains elusive. Several lines of evidence indicate that the scaffolding postsynaptic density (PSD) protein, PSD-95, is involved in orchestrating excitatory synapse maturation and specificity (7). PSD-95 is exclusively localized to glutamatergic synapses (8). Moreover, PSD-95 expression correlates with the period of excitatory synapse maturation (7, 9-11). Augmentation of excitatory synapse activity and ion channel clustering is driven by PSD-95 but not by related proteins, including synapse-associated protein (SAP)-102 and , and PSD-95 regulates clustering and activity of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors through a direct interaction with stargazin (7,(13)(14)(15)(16)(17)(18)(19)(20). However, it is unknown how PSD-95 effects are translated into changes in apposing presynaptic terminals. A candidate molecule for mediating PSD-95 effects on presynaptic maturation is the cell adhesion molecule neuroligin (NLG). NLG is present at excitatory postsynaptic sites and associates with the third PSD-95͞Dlg͞ZO-1 homology (PDZ) domain of PSD-95 through its C-terminal PDZ-binding site (2,21,22).The postsynaptic PDZ protein S-SCAM, another known binding partner of NLG, is also possibly involved in modulating NLG effects on ...

Nonsynaptic clusters of postsynaptic proteins have been documented; however, their role remains elusive. We monitored the trafficking of several candidate proteins implicated in synaptogenesis, when nonsynaptic clusters of scaffold proteins are most abundant. We find a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. A proportion of mobile clusters that exhibits slow movement and travels short distances contains neuroligin-1. The second group consists of stationary nonsynaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses.

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs.

ALS2 is an autosomal recessive form of spastic paraparesis (motor neuron disease) with juvenile onset and slow progression caused by loss of function of alsin, an activator of Rac1 and Rab5 small GTPases. To establish an animal model of ALS2 and derive insights into the pathogenesis of this illness, we have generated alsin-null mice. Cytosol from brains of Als2 ؊/؊ mice shows marked diminution of Rab5-dependent endosome fusion activity. Furthermore, primary neurons from Als2 ؊/؊ mice show a disturbance in endosomal transport of insulin-like growth factor 1 (IGF1) and BDNF receptors, whereas neuronal viability and endocytosis of transferrin and dextran seem unaltered. There is a significant decrease in the size of cortical motor neurons, and Als2 ؊/؊ mice are mildly hypoactive. Altered trophic receptor trafficking in neurons of Als2 ؊/؊ mice may underlie the histopathological and behavioral changes observed and the pathogenesis of ALS2.ALS ͉ alsin ͉ knockout mouse ͉ motor neuron ͉ Rab5

The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. However, factors that control this process remain unidentified. Here we show that members of the neuroligin (NLG) family, including NLG1, NLG2, and NLG3, drive the formation of both excitatory and inhibitory presynaptic contacts. The enrichment of endogenous NLG1 at excitatory contacts and NLG2 at inhibitory synapses supports an important in vivo role for these proteins in the development of both types of contacts. Immunocytochemical and electrophysiological analysis showed that the effects on excitatory and inhibitory synapses can be blocked by treatment with a fusion protein containing the extracellular domain of neurexin-1␤. We also found that overexpression of PSD-95, a postsynaptic binding partner of NLGs, resulted in a shift in the distribution of NLG2 from inhibitory to excitatory synapses. These findings reveal a critical role for NLGs and their synaptic partners in controlling the number of inhibitory and excitatory synapses. Furthermore, relative levels of PSD-95 alter the ratio of excitatory to inhibitory synaptic contacts by sequestering members of the NLG family to excitatory synapses.Synapse formation is a tightly regulated process that involves the recruitment of specific cell adhesion molecules and scaffolding proteins to newly formed contacts between an axon and a dendrite (1-3). In the brain, excitatory and inhibitory synaptic transmission is mainly mediated by two neurotransmitters: glutamate, which is released at excitatory glutamatergic synaptic contacts, and GABA, 1 which is released at inhibitory GABAergic synapses. Initial transformation of a contact to either an excitatory or inhibitory synapse is thought to be controlled by spatial and temporal changes in protein content. This process is critical because an appropriate balance between excitatory and inhibitory synapses is required for proper neuronal excitability and function (2, 4 -6). However, molecular events that control differentiation of a contact into either an excitatory or inhibitory synapse remain unknown.The postsynaptic density protein, PSD-95, is a molecule that is exclusively localized to glutamatergic synapses and regulates clustering of AMPA receptors through association with stargazin (7, 8). Through its third PDZ (PSD-95/Dlg/ZO-1) homology domain, PSD-95 also recruits neuroligin-1 (NLG1), a cell adhesion molecule involved in synapse formation (9 -11). These findings indicate that association of PSD-95 with NLG1 is involved in excitatory synapse development. Recent work by Prange et al. (12) showed that NLG1 can drive both excitatory and inhibitory presynaptic contact formation. These results suggested that NLGs are involved in inhibitory synapse formation. Our work also showed that the effects of NLG1 on postsynaptic differentiation were less dramatic. Overexpression of NLG1 modestly increased the total number of excitatory posts...

No abstract

Protein palmitoylation plays a critical role in sorting and targeting of several proteins to pre-and postsynaptic sites. In this study, we have analyzed the role of palmitoylation in trafficking of synaptotagmin I and its modulation by synaptic activity. We found that palmitoylation of N-terminal cysteines contributed to sorting of synaptotagmin I to an intracellular vesicular compartment at the presynaptic terminal. Presynaptic targeting is a unique feature of N-terminal sequences of synaptotagmin I because the palmitoylated N terminus of synaptotagmin VII failed to localize to presynaptic sites. We also found that palmitate was stably associated with both synaptotagmin I and SNAP-25 and that rapid neuronal depolarization did not affect palmitate turnover on these proteins. However, long-term treatment with drugs that either block synaptic activity or disrupt SNARE complex assembly modulated palmitoylation and accumulation of synaptotagmin I at presynaptic sites. We conclude that palmitoylation is involved in trafficking of specific elements involved in transmitter release and that distinct mechanisms regulate addition and removal of palmitate on select neuronal proteins.Synaptic transmission requires appropriate protein targeting and assembly of pre-and postsynaptic elements. Protein sorting to distinct domains in polarized cells appears to begin in the Golgi/trans-Golgi network, where proteins can segregate and exit in separate transport vesicles (1). One mechanism that regulates protein trafficking is palmitoylation, a post-translational modification involving the addition of palmitate, a 16-carbon fatty acid, via a labile thioester linkage (2-5). In neuronal cells, palmitoylation is critical for sorting of several synaptic proteins (6). These include the postsynaptic density protein PSD-95, the AMPA 1 receptor-binding protein, and the presynaptic proteins GAP-43 (growth-associated protein of 43 kDa) and GAD-65 (7-11). Palmitoylation of the AMPA receptor-binding protein and PSD-95 is essential for clustering at the PSD (8 -10), whereas palmitoylation of GAD-65 is important for presynaptic targeting (11,12).Acylation of several other axonal proteins as well as proteins associated with neurotransmitter release machinery has been recently reported (5, 6, 13). These include members of the synaptotagmin family that regulate synaptic vesicle trafficking and neurotransmitter release (14, 15). The synaptotagmin family includes 13 members characterized by a unique N-terminal region followed by a transmembrane domain, a cluster of cysteines (the putative palmitoylation site), a variable domain, and two C-terminal C2 domains (15-18). Synaptotagmin I, the most characterized member of the family, is proposed to act as a Ca 2ϩ sensor for regulated exocytosis (19). Other abundant members of the family include synaptotagmins III and VII (15). Interestingly, synaptotagmin I is localized to synaptic vesicles, whereas synaptotagmin VII is localized on the plasma membrane opposite synaptic vesicle docking sites (15). Despite th...