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. 2022 Oct 31;11:e80895. doi: 10.7554/eLife.80895

Figure 3. Phospho S270-insensitive DARPin-hFc 27G2 multiplexed with antibody Ab7a can assess synapse-specific gephyrin S270 phosphorylation.

(A) Representative images of DARPin-hFc 27G2 (but not antibody Ab7a) labeling gephyrin puncta in both wildype (WT) and phospho-mutant gephyrin S268A/S270A mutant mouse brain tissue (somatosensory cortex layer 2/3). (B) Representative images from hippocampal neuron culture showing the relative Ab7a signal (indicating S270 phosphorylation) varies by synapse and between neurons. (C) Representative image showing DARPin-hFc 27G2 binding at synaptic puncta in primary hippocampal neuron culture is preserved after inhibition of CDKs following 24 hr treatment with 5 µM aminopurvalanol (PurvA) while Ab7a staining is severely reduced. (D) The relative fluorescence intensity at individual synapses (pooled from 30 neurons per group) showing a pronounced decrease in the average Ab7a/DARPin-hFc 27G2 intensity ratio. Quantification of Ab7a/DARPin-hFc 27G2 fluorescence signal averaged across cells pooled from three independent experiments, n = 30 cells/group. (E) Representative images of hippocampal neuron culture used for quantification of relative Ab7a/DARPin-hFc labeling of clusters on the soma, proximal dendrites, or the axon-initial segment (A.I.S.) (AnkG). (F) Ab7a/DARPin intensity ratio of individual synapses pooled from 45 cells over three independent experiments showing a decrease in A.I.S. cluster Ab7a staining. Lower: quantification indicates significantly reduced A.I.S. Ab7a labeling of clusters compared to dendritic or somatic compartments. Statistics: (D) one-way ANOVA; (F) repeated-measures one-way ANOVA. All panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Mean and SD are presented.

Figure 3—source data 1. Values and statistical results used to generate Figure 3D and F.
elife-80895-fig3-data1.xlsx (301.2KB, xlsx)

Figure 3.

Figure 3—figure supplement 1. Structure of DARPin-hFc 27G2.

Figure 3—figure supplement 1.

DARPin clones were inserted into a construct containing an N-terminal HSA leader sequence for mammalian recombinant expression and a C-terminal hFc tag for detection with secondary antibodies.
Figure 3—figure supplement 2. Validation of DARPin-hFc 27G2 for immunostaining.

Figure 3—figure supplement 2.

Anti-gephyrin DARPin-hFc 27G2 labels postsynaptic gephyrin puncta in hippocampal neuron culture and adult brain tissue (layer 2/3 somatosensory cortex).
Figure 3—figure supplement 3. Competition with recombinant gephyrin reduces DARPin-hFc reactivity in tissue.

Figure 3—figure supplement 3.

DARPin-hFc 27G2 cluster detection is blocked by incubation with molar excess of recombinant gephyrin indicating its specificity in tissue.
Figure 3—figure supplement 4. Variation in Ab7a reactivity.

Figure 3—figure supplement 4.

The ratio of fluorescent intensity signal between pS270-specific antibody Ab7a and the phosphorylation nonspecific DARPin-hFc 27G2 indicates that Ab7a labeling is variable between clusters within and between individual synapses and neurons. Each data represents one cluster analyzed from six individual example neurons with different patterns of relative Ab7a reactivity. Median and SD are indicated in red.
Figure 3—figure supplement 4—source data 1. Values used to plot Figure 3—figure supplement 4.
Figure 3—figure supplement 5. Anti-gephyrin binders do not alter EGFP gephyrin cluster size.

Figure 3—figure supplement 5.

Embryonic E17 rat hippocampal neuron cultures were transfected with plasmid expressing EGFP-gephyrin at days in vitro (DIV) 8 and were fixed and stained using DARPin-hFc clones, antibody Ab7a, or no binder at DIV 15 and analyzed for EGFP-gephyrin cluster size along the principle dendrite. Median puncta size averaged by principal dendrite are presented from 6 to 10 neurons pooled across three independent experiments per group. Statistics: One-way ANOVA, n.s., no significant difference.
Figure 3—figure supplement 5—source data 1. Values and statistical analysis used to plot Figure 3—figure supplement 5.