(A) Fluorescence imaging of HRP-Mdga2, via BxxP labeling and neutravidin-AlexaFlour647 staining. Values give colocalization extent with inhibitory (vGAT) and excitatory (vGlut1) synapse markers analyzed from 8 fields of view each; errors, ± 1 s.d.; scale bar, 10 m. (B) Synaptogenesis assay based on overexpression of Nlgn2 to probe specificity of presynaptic vesicle recruitment. Nlgn2 was overexpressed along with Venus fluorescent protein (top), Venus-Mdga1 (middle), or Venus-Mdga2 (bottom). Two fields of view are shown per condition. Enhanced recruitment of excitatory or inhibitory synaptic vesicles to transfected neurons was assessed by staining with anti-vGlut1 and anti-vGAT antibodies, respectively. Images are representative of >20 transfected neurons per condition. Controls show that co-overexpression of Mdga1 or Mdga2 does not alter surface levels of V5-Nlgn2 (data not shown). (C) Quantitation of data in (B) along with 7 additional fields of view per condition. Synapse density is defined as total anti-vGlut1 or anti-vGAT intensity divided by area of transfected neuron. Errors, ± s.e.m. (D) Effect of Mdga1 or Mdga2 overexpression (without Nlgn2 co-overexpression) on excitatory and inhibitory vesicle densities, quantified as in (C). 12 fields of view analyzed per condition. Errors, ± s.e.m. (E) Effect of single or double knockdown of Mdga1 and/or Mdga2 on excitatory and inhibitory vesicle densities, quantified as in (C). Knockdowns (from 3 technical replicates per condition) verified by qPCR in (F) (errors, ± s.e.m.). Representative images in . 15 fields of view analyzed per condition. Errors, ± s.e.m. (G) Chimeras of MDGA1 and MDGA2 tested in (H). The parent genes each have 6 immunoglobulin (Ig) domains, a fibronectin type III (FNIII) domain, a memprin/A5 protein/receptor tyrosine phosphatase mu (MAM) domain, and a C-terminal GPI anchor. Chimera B exhibited poor surface trafficking and was not evaluated further. Numbers refer to amino acid residues at cross-over points. (H) Relative localization of chimeras to excitatory versus inhibitory synapses, assessed by imaging with anti-vGlut1 and anti-vGAT staining (images and error values shown in ). See also .