Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May 18:4:17.
doi: 10.1186/1749-8104-4-17.

Neuroligin1: a cell adhesion molecule that recruits PSD-95 and NMDA receptors by distinct mechanisms during synaptogenesis

Stephanie L Barrow  1 , John Rl Constable, Eliana Clark, Faten El-Sabeawy, A Kimberley McAllister, Philip Washbourne
Affiliations

Neuroligin1: a cell adhesion molecule that recruits PSD-95 and NMDA receptors by distinct mechanisms during synaptogenesis

Stephanie L Barrow et al. Neural Dev. .

Abstract

Background: The cell adhesion molecule pair neuroligin1 (Nlg1) and beta-neurexin (beta-NRX) is a powerful inducer of postsynaptic differentiation of glutamatergic synapses in vitro. Because Nlg1 induces accumulation of two essential components of the postsynaptic density (PSD) - PSD-95 and NMDA receptors (NMDARs) - and can physically bind PSD-95 and NMDARs at mature synapses, it has been proposed that Nlg1 recruits NMDARs to synapses through its interaction with PSD-95. However, PSD-95 and NMDARs are recruited to nascent synapses independently and it is not known if Nlg1 accumulates at synapses before these PSD proteins. Here, we investigate how a single type of cell adhesion molecule can recruit multiple types of synaptic proteins to new synapses with distinct mechanisms and time courses.

Results: Nlg1 was present in young cortical neurons in two distinct pools before synaptogenesis, diffuse and clustered. Time-lapse imaging revealed that the diffuse Nlg1 aggregated at, and the clustered Nlg1 moved to, sites of axodendritic contact with a rapid time course. Using a patching assay that artificially induced clusters of Nlg, the time course and mechanisms of recruitment of PSD-95 and NMDARs to those Nlg clusters were characterized. Patching Nlg induced clustering of PSD-95 via a slow palmitoylation-dependent step. In contrast, NMDARs directly associated with clusters of Nlg1 during trafficking. Nlg1 and NMDARs were highly colocalized in dendrites before synaptogenesis and they became enriched with a similar time course at synapses with age. Patching of Nlg1 dramatically decreased the mobility of NMDAR transport packets. Finally, Nlg1 was biochemically associated with NMDAR transport packets, presumably through binding of NMDARs to MAGUK proteins that, in turn, bind Nlg1. This interaction was essential for colocalization and co-transport of Nlg1 with NMDARs.

Conclusion: Our results suggest that axodendritic contact leads to rapid accumulation of Nlg1, recruitment of NMDARs co-transported with Nlg1 soon thereafter, followed by a slower, independent recruitment of PSD-95 to those nascent synapses.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Nlg1 clusters were mobile in dendrites of cortical neurons. (A) Image of a live neuron transfected with GFP-Nlg1 (green) and surface-labeled with anti-GFP Fab fragment (red). The arrowhead denotes one of the few clusters of GFP-Nlg1 that was not labeled by the Fab fragment. Scale bar, 5 μm. (B) Plot of the fluorescence intensities (in arbitrary units (a.u.)) for GFP-Nlg1 and anti-GFP Fab fragment from five neurons, showing strong correlation of intensities. (C) Time-lapse imaging of 4 d.i.v. neurons transfected with CFP-Nlg1 revealed Nlg1 clusters that were mobile in the dendrites (pink and black arrowheads) and filopodia (arrow). Immobile clusters are indicated by asterisks in the first panel. Time is in minutes and seconds. Scale bar, 10 μm. (D) Diagram depicting the principle interaction domains of Nlg1 and the deletions of the carboxy-terminal 4 and 55 amino acids (ΔC4, ΔC55). AChE, acetylcholinesterase domain; PDZb, PDZ binding motif; WW, ww interaction domain; Wt, wild type. (E) Images of live transfected neurons demonstrated the punctate distribution of GFP-Nlg1 (wild type (wt)) and GFP-Nlg1ΔC4 (ΔC4). The localization of GFP-Nlg1ΔC55 (ΔC55) was diffuse and showed very few clusters.
Figure 2
Figure 2
Nlg1 accumulated at, and was transported to, sites of axodendritic contact. (A) Formation of a contact between 5 d.i.v. neurons transfected with GFP-Nlg1 (green) and mCherry (red). Time 0:00 is defined as the first time of stabilized contact between the dendrite and axonal growth cone. Nlg1 accumulated at this site of contact within 3 minutes. Higher magnification images of the region of contact (white box) are shown to the right and with fluorescent intensity plots for both the dendritic (green) and axonal (red) region. (B) Fluorescence intensity (F) changes in arbitrary units (a.u.), for examples of rapid (left) and slower accumulation of Nlg1 (right) following contact. Black traces are taken from a dendritic segment contacted by axonal growth cones and grey traces are taken from a control dendritic segment, away from the site of contact. The gray line at time 0 represents the time of contact. Blank portions of the right trace are measurements that have been deleted due to focal drift. (C) Continuation of imaging of the GFP-Nlg1 transfected neuron shown in (A). Extension of a filopodium from the main dendritic branch at the site of the initial Nlg1 accumulation occurred 30 minutes following contact between the growth cone and the dendrite. Accumulation of Nlg1 in the dendrite at the site of contact (black arrowhead) clustered in the filopodial tip, which extended out over the axonal growth cone (axon shown as a dotted outline). A Nlg1 cluster remained at the tip of the filopodium during extension (open arrowhead). (D) Images of neighboring neurons transfected with GFP-Nlg1 (green) and mCherry (red). At time point 0:00 the axonal growth cone had already made contact with the dendrite. A cluster of Nlg1 (white arrowhead) moved to that pre-formed contact and remained stable. A stationary cluster of Nlg1, present at a second pre-existing contact site (top of image), also remained stable for the duration of imaging. Time is in minutes and seconds and the scale bars are 5 μm in all images.
Figure 3
Figure 3
Patching of endogenous and recombinant Nlg. (A) Diagram depicting the two methods of patching: pre-oligomerized β-NRX-Fc fusion proteins were used to patch endogenous Nlg and anti-GFP antibodies to patch recombinant GFP-Nlg1. (B) Fluorescence image of live cortical neurons demonstrating β-NRX-Fc bound to non-transfected neurons (asterisks indicate cell bodies). Scale bar, 20 μm. (C) Confocal images of cortical neurons transfected with GFP-Nlg1 and immunostained with antibodies against GFP (green) and human Fc (blue) after 0, 5 and 60 minutes of patching with β-NRX-Fc. Scale bar, 20 μm. (D) Quantification of the time course of patching for both β-NRX-Fc clusters and GFP-Nlg1 clusters per 20 μm of dendrite (n ≥ 13 neurons for each time point).
Figure 4
Figure 4
Patching endogenous Nlg induced clustering of PSD-95. (A) Patching with β-NRX-Fc (patched) increased the density of PSD-95 clusters as shown by confocal images of neurons that were incubated with β-NRX-Fc (red) for 1 hour then fixed and immunostained for PSD-95 (green). Secondary antibody only was used as the control. Scale bar, 20 μm. (B, C) Enlargements of the boxed regions in (A), showing control and patched dendrites, respectively. Scale bar, 10 μm. (D) Time-course of PSD-95 clustering by β-NRX-Fc demonstrates that the increase in PSD-95 cluster density occurred after 30 minutes of patching. (E) The density of PSD-95 clusters (per 20 μm of dendrite) was increased by patching Nlg, but not in the presence of 2-bromopalmitate (*P < 0.004). (F) Live imaging of a cortical neuron during patching with β-NRX-Fc demonstrates the appearance of new PSD-95-GFP clusters (yellow arrows). Sites at which clusters appeared are denoted with ghost arrows in the first image. Time is in hours and minutes. Scale bar, 5 μm. (G) Time-course of fluorescence intensities from five neurons treated with β-NRX-Fc. The locations at which intensities were measured have been categorized into sites with stable fluorescence (constant), sites at which clusters appear (appear) and sites at which clusters disappear (disappear). (H) Analysis of the numbers of clusters per neuron as categorized in (G). Error bars are standard error.
Figure 5
Figure 5
Patching of GFP-Nlg1 induced clustering of PSD-95. (A) Patching of GFP-Nlg1 with anti-GFP (green) increased the number of PSD-95 clusters in the dendrites of transfected cortical neurons (red). 'True' synapses are defined as colocalized VGlut1 (blue) and PSD-95 (red) protein clusters. Scale bar, 20 μm. (B) The density of non-synaptic PSD-95 clusters colocalized with Nlg1 is increased by patching recombinant GFP-Nlg1 at 60 minutes. (C) When synaptic clusters are excluded from analysis (by eliminating clusters that are positive for VGlut1), it is clear that the increase in PSD-95 clusters when patched with anti-GFP antibody was due to accumulation at non-synaptic sites. This clustering of PSD-95 was blocked when the carboxy-terminal region is deleted in either ΔC4 or ΔC55 mutants (*P < 0.02). Error bars are standard error.
Figure 6
Figure 6
Patching Nlg1 did not affect non-synaptic NMDA receptor distribution. (A) The localization and density of NR1 clusters (red) was not altered by patching of transfected GFP-Nlg1 (green). NR1 was colocalized with GFP-Nlg1 clusters before and after patching (arrowheads). Scale bar, 10 μm. (B) Patching with β-NRX-Fc (red) also did not alter the already high colocalization of Nlg with NR1 (green). Scale bar, 5 μm. (C) Patching with β-NRX-Fc did not significantly change the density of non-synaptic NR1 clusters in cortical neurons or neurons treated with 2-bromopalmitate. (D) Patching of Nlg1 increased the intensity of NR1 at synaptic sites (*P < 0.05) and this increase was prevented by 2-bromopalmitate. Error bars are standard error.
Figure 7
Figure 7
Colocalization of Nlg1 and NMDA receptors prior to synapse formation. (A) Immunostaining of cortical neurons in culture at 4 and 10 d.i.v. with antibodies to Nlg1 (red), NR2A/B(green) and VGlut1 (blue). At 4 d.i.v., the majority of colocalized clusters of Nlg1 and NR2A/B were non-synaptic (arrowheads), whereas in 10 d.i.v. neurons the majority of colocalized NR2A/B and Nlg1 were found with the presynaptic marker VGlut1 (arrows). (B) The density of protein clusters as indicated in the legend are plotted over time in culture. Nlg/NR2 includes the clusters of NR2 (green circles) and Nlg1 (red squares) that colocalize with each other. The rest of the cluster densities in (B, C) are plotted similarly. Clusters of colocalized NR2A/B and Nlg1 were abundant at early time points in culture. The density of non-synaptic (ns) Nlg/NR2 positive clusters decreased with time. (C) The densities of presynaptic terminals and presynaptic terminals with Nlg1 and NR2 increased steadily over time in culture. Error bars are standard error. *P < 0.05, **P < 0.001; asterisks at the end of the curve refer to comparisons between 4 and 15 d.i.v.
Figure 8
Figure 8
NMDA receptor transport packets (NRTPs) and Nlg1 clusters were co-transported prior to synaptogenesis. (A) Time-lapse imaging of neurons cotransfected with CFP-Nlg1 (green) and NR1-DsRed (red) demonstrates movement of CFP-Nlg1 with NR1-DsRed (arrowheads) in both the anterograde and then retrograde direction. In the first panel, CFP-Nlg1 was seen at the tips of filopodia (arrows). Lower panels are enlargements of the boxed region in the upper panel. Time is in minutes and seconds. Scale bar, 10 μm. (B) Time-lapse images demonstrate that CFP-Nlg1 can move with (arrowheads) and without (arrows) NR1-DsRed. Grey arrows and arrowheads indicate original locations of clusters from first image. Scale bar, 10 μm. (C) CFP-Nlg1 clusters had higher mean velocities than NRTPs or co-transported NR1 and Nlg1. Error bars are standard error. Only mobile clusters were included in this analysis (*P < 0.02). (D) Comparison of the distribution of mean velocities of mobile clusters of Nlg alone (green) and colocalized CFP-Nlg1 and NR1-DsRed (yellow).
Figure 9
Figure 9
Transient associations of Nlg and NMDA receptor transport packets (NRTPs) and the effects of Nlg patching on NRTP mobility. (A) Time-lapse imaging of 4 d.i.v. neurons cotransfected with NR1-DsRed (red) and CFP-Nlg1 (green) demonstrates that NRTPs (arrowheads) can move between stable Nlg clusters. Grey arrowheads indicate original location of cluster. Clusters that were immobile during the imaging period are indicated by an asterisk in the top panel. Time is in minutes and seconds. Scale bar, 5 μm. (B) Alternatively, CFP-Nlg1 can move to a stable NR1-DsRed cluster and then the two can move together (arrowheads). Clusters that were immobile during the imaging period are indicated by an asterisk in the top panel. Scale bar, 5 μm. (C) Time-lapse imaging of neurons co-transfected with CFP-Nlg1 (green) and NR1-DsRed (red) after patching with β-NRX-Fc for at least 1 hour. Over 65% of NR1-DsRed was colocalized with CFP-Nlg1 (white arrowheads). One cluster of NR1-DsRed alone is highlighted with a yellow arrowhead. Scale bar, 10 μm. (D) The percentage of total NR1-DsRed clusters that were mobile was significantly decreased after patching with β-NRX-Fc (numbers of neurons analyzed are indicated above bars). Error bars are standard error.
Figure 10
Figure 10
Biochemical analysis of the interaction between Nlg and NMDA receptor-containing immuno-isolates. (A) NR2B and Nlg were immunoprecipitated from detergent solubilized P4 rat visual cortex and blotted with antibodies specific for NR1 and Nlg1. NR1 and Nlg1 were co-immunoprecipitated using these antibodies, but not by Protein-G sepharose beads alone (control). (B) Immunoprecipitations (IPs) from detergent-solubilized P4 rat visual cortex using antibodies to NR2B and Nlg did not co-immunoprecipitate the synaptic proteins synapsin1 and GluR2. (C) NR1-containing membrane fractions were immuno-isolated from P2–3 rat cortex. Western blots of these organelles reveal enrichment for many other postsynaptic proteins, including Nlgs 1, 2 and 3. Input and control lanes were included for comparison. Control lanes represent isolations performed without primary antibody to NR1 (beads were coated with control IgG). Comparison of NR1 and control lanes shows enrichment of the blotted protein in NMDA receptor transport packets (NRTPs). (D) Nlg1 remained associated with NR1 in the immuno-isolated vesicle fraction after solubilization with Triton X-100, while GluR2 is no longer recovered. (E) Nlg1 was not enriched after washing with a carbonate buffer at pH11, which removes associated, but not integral vesicle proteins. In comparison, GluR2 remains associated with NRTPs under high pH conditions.
Figure 11
Figure 11
Nlg PDZ binding motif is important for co-transport of Nlg1 and NMDA receptors. (A) Time-lapse imaging of neurons co-transfected with either GFP-Nlg1 (green, left panel) and NR1-DsRed (red), or GFP-NlgΔC4 (green, right panel) and NR1-DsRed (red). Arrowheads indicate colocalized Nlg1 and NR1 clusters. Scale bar, 5 μm. (B) Fluorescence intensity profiles (in arbitrary units (a.u.)) along a line drawn through the dendrite, between the white faded lines shown in (A, top panels). Boxed regions around fluorescent intensity peaks correspond to colocalization of Nlg and NR1. (C) Deletion of the PDZ binding motif within the cytoplasmic tail of Nlg significantly reduced the colocalization of total and mobile Nlg clusters with NR1. Error bars are standard error; *P ≤ 0.001.
Figure 12
Figure 12
Model of the association between Nlg and NMDA receptor transport packets (NRTPs) during transport. NRTPs cycle with the plasma membrane (pale arrows) during pauses of microtubule-dependent transport (yellow arrows). These can be associated with clusters of Nlg (red) at the membrane both during transport and pausing. The interaction between surface Nlg and NRTPs is likely via membrane-associated guanylate kinases (orange beads on a string).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McAllister AK. Dynamic aspects of CNS synapse formation. Annu Rev Neurosci. 2007;30:425–450. doi: 10.1146/annurev.neuro.29.051605.112830. - DOI - PMC - PubMed
    1. Ziv NE, Garner CC. Principles of glutamatergic synapse formation: seeing the forest for the trees. Curr Opin Neurobiol. 2001;11:536–543. doi: 10.1016/S0959-4388(00)00246-4. - DOI - PubMed
    1. Ahmari SE, Buchanan J, Smith SJ. Assembly of presynaptic active zones from cytoplasmic transport packets. Nat Neurosci. 2000;3:445–451. doi: 10.1038/74814. - DOI - PubMed
    1. Waites CL, Craig AM, Garner CC. Mechanisms of vertebrate synaptogenesis. Annu Rev Neurosci. 2005;28:251–274. doi: 10.1146/annurev.neuro.27.070203.144336. - DOI - PubMed
    1. Scheiffele P. Cell-cell signaling during synapse formation in the CNS. Annu Rev Neurosci. 2003;26:485–508. doi: 10.1146/annurev.neuro.26.043002.094940. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

-