To quantify the synaptic puncta size, we thresholded all the images equally and measured the average pixel intensities along the dendritic segments in the transfected neurons by manually tracing each puncta with two-fold background transmission. dimerization was not required for either the non-neuronal or the neuronal synapse-formation assay. Nevertheless, both -neurexin binding and neuroligin-1 dimerization were essential for the increase in apparent synapse size that is induced by neuroligin-1 in transfected neurons. Thus, neuroligin-1 performs diverse synaptic functions by mechanisms that include as essential components of -neurexin binding and neuroligin dimerization, but lengthen beyond these activities. (2007)NL1C1D242N/Q243A/K246AERNDNDNDSBAThis studyNL1C2Q256A/R259AERNDNDNDSBAThis KT182 studyNL1C3Q328A/Y332APM++NDSBAThis studyNL1C4R371A/Y372A/H373APM++NDSBAThis studyNL1C5L399A/N400A/D402NPM?+ 10 MSBAArac (2007)NL1C6D421N/D423N/D424NPM++NDSBAThis studyNL1C7D440N/N441A/D424NPM++NDSBAThis studyNL1C8N468A/E470A/R472A/K474AERNDNDNDSBAThis studyNL1C9E542Q/L543A/F544AERNDNDNDSBAThis studyNL1C10F582A/K586A/R589APM++NDSBAThis studyNL1C11E128Q/V129APM++NDSBAThis studyNL1C21E72Q/I73APM++NDSBAThis studyNL1C22D140NPM++NDSBAThis studyNL1C23H294A/E297AERNDNDNDSBAThis studyNL1C24L302A/S321A/S322AERNDNDNDSBAThis studyNL1C25Y509A/H511A/Q513APM++NDSBAThis studyNL1C31L399A/N400A/D402N/K306APM?+NDSBAThis studyNL1C32L399A/N400A/D402N/E297A/K306APM?? 10 MSPR/SBAArac (2007)NL1C33L399A/N400A/D402N/L273APM?+ND.SBAThis studyNL1C34L399A/N400A/D402N/K601A/D602APM?+NDSBAThis studyNL1C35L399A/N400A/D402N/Q395A/E397APM?? 50 MSPR/SBAArac (2007)NL1C36L399A/N400A/D402N/D571A/Q574AERNDNDNDSBAThis studyNL1C37L399A/N400A/D402N/F499APM??NDSBAThis studyNL1C38T111A/Q112AERNDNDNDSBAThis studyNL1C39E92A/H93APM++NDSBAThis studyNL1C40F458A/W463APM++NDSBAThis studyNL1C41Q147A/D148APM++NDSBAThis studyNL1C42R63A/K66AERNDNDNDSBAThis studyNL1C43S102A/S105APM++NDSBAThis studyNL1C44F582A/K586A/R589A/E72Q/I73APM++NDSBAThis studyNL1C45Y509A/H511A/Q513A/D421N/D423N/D424NPM++NDSBAThis KT182 studyNL1C46Q328A/Y332A/D440N/N441A/Y445APM++NDSBAThis studyNL1C51F458A/M459A/W463APM++NDSBAThis studyNL1C53F458A/M459A/W463A L629A/L633APM++NDSBAThis studyNL1C54F458A/M459A/W463A/L399A/N400A/ D402N/E297A/K306AERNDNDNDNDThis studyNL1CCT776 followed by stop codonPM++NDSBAThis studyER, endoplasmic reticulum; ITC, isothermal titration calorimetry; ND, not decided; NL1, neuroligin-1; PM, plasma membrane; SPR, surface plasmon resonance; SBA, surface binding assay (Figures 1, Supplementary Physique S4 and S5); all neuroligin-1 molecules used lacked inserts in splice sites A and B, and contained a cytoplasmic monomeric mVenus tag inserted between residues 776 and 777. Open in a separate windows We first examined the expression levels and surface transport of all neuroligin-1 mutants. These measurements were intended to eliminate misfolded neuroligin-1 mutants that are likely to be poorly expressed, and unlikely to reach the cell surface (Comoletti (2007). Again, the neurexin binding or dimerization mutations experienced no effect on the increase in synaptic strength or NMDA/AMPA ratio induced by overexpressed neuroligin-1 (Physique 7). Open in a separate window Physique 7 Electrophysiological effects of neuroligin-1 mutants in transfected neurons. Neurons were transfected with mVenus alone (Control), with wild-type neuroligin-1 (NL1 WT), numerous neurexin-binding mutants of neuroligin-1 (NL1C5, C32, and C35), or the NL1C51 dimerization-defective mutant of neuroligin-1. (A) Representative traces (left) and bar diagrams of the frequency (centre) and amplitude (right) of miniature EPSCs. (B) Representative traces (left) and diagram bars of the amplitude (right) of action-potential-evoked AMPAR-dependent EPSCs. (C) Representative traces of NMDA- and AMPA-receptor-dependent EPSCs (left), and bar diagrams of the measured NMDA/AMPA-receptor EPSC ratio (right). (D) Representative traces (left) and diagram bars of the amplitude (right) of action-potential-evoked AMPAR-dependent EPSCs. (E) Representative traces (left) and mean charge transfer (right, integrated over 30 s) of EPSCs elicited by hypertonic sucrose (0.5 M sucrose for 30 s). (F) Representative traces (left) and diagram bars of the frequency (centre) and amplitude (right) of miniature IPSCs in neurons expressing mVenus, wild-type neuroligin-1 (NL1 WT), or NL1C32 neurexin-binding mutant of neuroligin-1 (NL1C32). (G) Representative traces (left) and diagram bars of the amplitude (right) of action-potential-evoked IPSCs. Recordings were from your transfected neurons recognized by mVenus fluorescence. The level bars apply to all traces in a set. Data shown are meanss.e.m. Asterisks above the bar diagrams indicate statistically significant differences in pairwise comparisons between the control KT182 and KT182 experimental groups ( em n /em =3C8 impartial experiments; em *P /em 0.05, em **P /em 0.01). Dashed and dotted lines refer to the values of mVenus as a negative control. To investigate whether the increased evoked EPSC after neuroligin-1 overexpression is because of an increase in release probability or in the readily releasable pool (RRP), we measured the paired-pulse ratio of closely spaced EPSCs (as an indirect measure of release probability), and the size of the EPSC induced by application of hypertonic sucrose (which triggers synaptic-vesicle exocytosis in a Ca2+-impartial manner and is thought to allow measurements of the entire RRP of the synapses on a neuron; Rosenmund and Stevens, 1996). We found that neuroligin-1 overexpressionwild-type or mutantenhanced the RRP (Physique 7E), but did not switch the paired-pulse ratio (Supplementary Physique S8). The effects of wild-type and NL1C32 mutant neuroligin-1 around the RRP and on action-potential-evoked EPSCs were similar. These results suggest that neuroligin-1 increases the overall synaptic capacity around the neuron, despite its lack of an effect around the spontaneous miniature release rate, by a mechanism that is impartial of neurexin binding. These results also suggest that the observed neurexin-dependent increase in apparent synapse size does not alter the global physiological properties of the synapses on a neuron. Conversation Multiple recent studies showed that neuroligin-1 is usually a postsynaptic cell-adhesion molecule that has an important function in shaping central synapses in KT182 brain, and that neuroligin-1 acts by binding to neurexins (e.g. Track em et al /em , 1999; Dean em et al /em , 2003; Varoqueaux em et al /em , 2006; Chubykin em et al /em , 2007). Moreover, neuroligin-1 forms constitutive homodimers (Comoletti em et al /em , 2003), leading to the postulate that its transmission is usually transduced through dimerization of neurexins (Dean em et al /em , 2003). Mela The goal of this study was to directly test these hypotheses..
