The Journal of Neuroscience, April 19, 2006, ():
Structure Function and Splice Site Analysis of the Synaptogenic Activity of the Neurexin-1
LNS Domain
J. Neurosci. Graf et al.
26: 4256
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. Surface localization of mutated myc-neurexin-CFP proteins. (A-L) Wild type and mutated neurexin proteins with extracellular myc tags and intracellular CFP tags were transfected into COS cells and visualized by CFP and live cell myc staining. Unmutated nrx(-S4) (A) and nrx(+S4) (B) reached the cell surface at similar levels as the mutated proteins β2β3mid (C), β2β3end (D), β3β4 (E), β4β5 (F), β6β7 (G), β10β11 (J), β11β12 (K), and α1β13 (L) as determined by myc staining. The absence of myc staining suggests that proteins with mutations in the β7β8 (H) and β9β10 (I) loops failed to reach the cell surface. (M) A structural model of the neurexin LNS domain (from Rudenko et al., 1999) shows the mutated loop regions in red. β-strands are visualized in yellow and the α-helix is orange. The model is of the splice insert-negative variant, although the location of 30 aa insertion at S4 in the β10β11 loop is noted. Scale bar 20 μm.
- supplemental material
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Supplementary Figure 2. Neurexin mutations that reduce synaptogenic activity reduce recruitment of neuroligins-1/3/4 and -2. (A-D) In a fibroblast-neuron coculture assay, COS cells expressing CFP-tagged neurexin-1β mutants T108A (A,C) or T205A (B,D) induced clustering of both neuroligin-1/3/4 (A,B) and neuroligin-2 (C,D) in contacting dendrites. Induced clusters (arrowheads) were negative for the presynaptic antigen synapsin unlike endogenous synapses. However, the extent of clustering appeared reduced for these mutants, especially for T205A, compared with wild type neurexin-1β (compare with Fig. 2A,D). (E) Quantification of the total integrated intensity of synapsin-negative neuroligin-1/3/4 and -2 clusters underlying transfected fibroblasts revealed reduced induction of neuroligin-1/3/4 and -2 clusters compared to wild type nrx(-S4) (* p < 0.05, ** p<0.001, n = 20-40 cells each). Scale bar 10 μm.
