Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We imaged receptor fields at ultra-high resolution at freeze-fractured membranes, tracked surface and internalized glycine receptors (GlyR) and studied differential regulations of GlyRβ interactions with the scaffold protein gephyrin and the F-BAR domain protein syndapin I and thereby reveal key principles of this process. S403 phosphorylation of GlyRβ, known to be triggered by synaptic signaling, caused a decoupling from gephyrin scaffolds but simultaneously promoted association of syndapin I with GlyRβ. In line, kainate-treatments used to trigger rearrangements of glycine receptors in murine syndapin I KO spinal cords (mixed sex) showed even more severe receptor field fragmentation than already observed in untreated syndapin I KO spinal cords. Syndapin I KO furthermore resulted in more dispersed receptors and increased receptor mobility also pointing out an important contribution of syndapin I in the organization of GlyRβ fields. Strikingly, syndapin I KO also led to a complete disruption of kainate-induced GlyRβ internalization. Accompanying quantitative ultra-high resolution studies in dissociated spinal cord neurons strongly suggested that the observed defects in GlyR internalization observed in syndapin I KO spinal cords are directly caused by syndapin I deficiency within murine spinal cord neurons. Together our results unveiled important mechanisms organizing and altering glycine receptor fields during both steady-state and particularly upon kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy and plasticity of glycinergic synapses in the spinal cord.SIGNIFICANCE STATEMENTInitial observations suggested that also glycinergic synapses - key for spinal cord and brain stem functions - may display some form of synaptic plasticity. Imaging receptor fields at ultra-high resolution at freeze-fractured membranes, tracking surface and internalized glycine receptors (GlyR) and studying regulations of GlyRβ interactions we here reveal key principles of these kainate-inducible adaptations. A switch from gephyrin-mediated receptor scaffolding to syndapin I-mediated GlyRβ scaffolding and internalization allows for modulating synaptic receptor availability. In line, kainate-induced GlyRβ internalization was completely disrupted and GlyRβ receptor fields were distorted upon syndapin I KO. These results unveiled important mechanisms during both steady-state and kainate-induced alterations of synaptic GlyR fields - principles underlying synaptic efficacy and plasticity of synapses in the spinal cord.

The coordinated action of a plethora of factors is required for the organization and dynamics of membranous structures critically underlying the development and function of cells, organs, and organisms. The evolutionary acquisition of additional amino acid motifs allows for expansion and/or specification of protein functions. We identify a thus far unrecognized motif specific for chordata EHBP1 proteins and demonstrate that this motif is critically required for interaction with syndapin I, an F-BAR domain-containing, membrane-shaping protein predominantly expressed in neurons. Gain-of-function and loss-of-function studies in rat primary hippocampal neurons (of mixed sexes) unraveled that EHBP1 has an important role in neuromorphogenesis. Surprisingly, our analyses uncovered that this newly identified function of EHBP1 did not require the domain responsible for Rab GTPase binding but was strictly dependent on EHBP1's syndapin I binding interface and on the presence of syndapin I in the developing neurons. These findings were underscored by temporally and spatially remarkable overlapping dynamics of EHBP1 and syndapin I at nascent dendritic branch sites. In addition, rescue experiments demonstrated the necessity of two additional EHBP1 domains for dendritic arborization, the C2 and CH domains. Importantly, the additionally uncovered critical involvement of the actin nucleator Cobl in EHBP1 functions suggested that not only static association with F-actin via EHBP1's CH domain is important for dendritic arbor formation but also actin nucleation. Syndapin interactions organize ternary protein complexes composed of EHBP1, syndapin I, and Cobl, and our functional data show that only together these factors give rise to proper cell shape during neuronal development.