Supplemental Material
DNA constructs
Expression constructs for HA-tagged mouse NL1(-), NL1B, NL1AB and NL2A splice forms were previously described (Scheiffele et al., 2000). NL1A was constructed by sub-cloning an NheI - EcoNI fragment containing the A insertion of NL1AB into NL1(-). All NL1 splice forms were transferred to an expression vector containing the chicken beta actin promoter (pCAGGS). NL2(-) sequences were subcloned from cDNA clone GI 56699424 (distributed by Invitrogen) using AleI and PflMI restriction sites. To insert the NL1 splice site B sequences into NL2, overlap extension PCR was performed with the following primers: NL2Age1, 5’-AAGAAACCGGTCATGCTGTTTCTA-3’, NL2EcorI, 5’-TGAGGAATTCCCCCTGTTGCATGA-3’, NL2Binsert1, 5’-GGTTGAATTGCTCCAACGGTTACCTTCTGAGTGGTGGGAAAGGAT-3’
NL2Binsert2, 5’-AACCGTTGGAGCAATTCAACCAAAGGACTGTTCCAGAAGGCCATT-3’. The final PCR product was digested with restriction enzymes, AgeI and EcoRI and sub-cloned into the NL2(-) or NL2A plasmid, respectively. The mutant NL1AB N303A was generated using a similar overlap extension strategy with the following primers: NL1EcoN1, 5’-CCTCATCCAGGCCCTAAGATG-3’, NL1N303A1, 5’-GTCCTTTGGTTGAAGCGCTCCAACGGTTACC-3’, NL1N303A2, 5’-GGTAACCGTTGGAGCGCTTCAACCAAAGGAC-3’, NL1PvuII, 5’-TCTACTAACTCTACTGTATCT-3’. Full length expression constructs for mouse NRX1b4(-), and NRX1b4(+) were previously described (Dean et al., 2003). For HA-tagged rat NRX1a4(-), a full length cDNA was isolated from adult rat RNA. The HA-epitope was inserted at the mature N-terminus behind the signal sequence. All DNA constructs were verified by sequencing.
Primers for RT-PCR analysis of NL1 and NL2 splice variants were: NL1A, 5’-CTATACTTAAACATCTATGTCCCA-3’ and 5’-GTAGCTTGCCAACACACTCCCATCAT-3’; NL1B, 5’-GCTGACTTTATCCCATTATTCTGAA-3’ and 5’-GCTGGAAAGGGCTGTTCCACTCTGA-3’; NL2A, 5’-CTGTACCTCAACCTCTACGTGCCC-3’ and 5’-ATAGGCAGCCAGGACTGAGCCGTCG-3’.
Supplemental Figures:
Figure S1. Size of presynaptic vesicle clusters induced by neuroligin-1 over-expression.
Cumulative distribution of VGlut1- and VGAT-positive puncta in hippocampal neurons over-expressing EGFP, NL1(-), or NL1B. Sizes of individual puncta were grouped into bins of 50 pixel increments (>250 puncta total, from 10 cells each).
Figure S2: Endogenous neurexins are recruited to glutamatergic and GABAergic synaptic sites in response to over-expression of NL1AB or NL2A. Hippocampal neurons 10 DIV were transfected with neuroligin isoforms for 3 days. Cultures were immunostained with anti-HA antibodies to detect neuroligin isoforms (green), pan-neurexin antibodies (red), and antibodies to presynaptic markers VGlut1 or VGAT (blue). Scale bar is 5 µm.
Figure S3: Differential binding of NRX1b4(+) to neuroligin splice variants.
(A) COS cells expressing neuroligin isoforms were incubated with a control IgG or purified recombinant Fc-fusion proteins of NRX1b4(-) or NRX1b4(+). Panels show merged signals for the HA-tagged neuroligin isoforms (green) and the bound Fc proteins (red) and single channel images of the Fc-proteins. Scale bar is 10 µm.
(B) For quantification, the average intensity of anti-HA-reactivity (detecting the transfected NL isoform) and Fc-immunereactivity was measured. The intensity ratio (FC/ HA) for control FC is in black, NRX1b4(-)Fc is in blue, and NRX1b4(+) is in red (mean ± SD). The control Fc protein did not bind to any neuroligin isoforms, while NRX1b4(-)Fc bound to all neuroligin isoforms. NRX1b4(+)Fc bound to a subset of neuroligin isoforms: NL1(-), NL1A, NL2(-) and NL2A).
Figure S4: Recruitment of endogenous NL2 to NRX1b4(+) and NRX1a4(-) – induced postsynaptic structures. Mixed cultures of COS cells expressing NRX1b4(+) and NRX1a4(-) were immunostained with antibodies to the HA-epitope in the neurexin isoform (blue), NL2-specific antibodies (red), and antibodies to gephyrin (green). Scale bar is 10 µm.
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