The presynaptic active zone mediates synaptic vesicle exocytosis, and modulation of its molecular composition is important for many types of synaptic plasticity. Here, we identify synaptic scaffold protein liprin-α2 as a key organizer in this process. We show that liprin-α2 levels were regulated by synaptic activity and the ubiquitin-proteasome system. Furthermore, liprin-α2 organized presynaptic ultrastructure and controlled synaptic output by regulating synaptic vesicle pool size. The presence of liprin-α2 at presynaptic sites did not depend on other active zone scaffolding proteins but was critical for recruitment of several components of the release machinery, including RIM1 and CASK. Fluorescence recovery after photobleaching showed that depletion of liprin-α2 resulted in reduced turnover of RIM1 and CASK at presynaptic terminals, suggesting that liprin-α2 promotes dynamic scaffolding for molecular complexes that facilitate synaptic vesicle release. Therefore, liprin-α2 plays an important role in maintaining active zone dynamics to modulate synaptic efficacy in response to changes in network activity.

Liprin-α proteins are major protein constituents of synapses and are important for the organization of synaptic vesicles and neurotransmitter receptors on their respective sides of the synapse. Although it is becoming apparent that the single liprin-α gene in invertebrates is essential for synapse function, it is not known to what extent the four different liprin-α homologs (liprin-α1-4) in mammals are involved at synapses. We have designed specific antibodies against each of the four liprin-α proteins and investigated their regional and cellular distribution in the brain. Here we show that all four liprin-α proteins are present throughout the mature brain but have different regional distributions, which is highlighted by their differential localization in olfactory bulb, hippocampus, and cerebellar cortex. Double-immunofluorescence staining indicates that different liprin-α proteins are enriched in different synaptic populations but are also present at nonsynaptic sites. In particular, liprin-α2 is preferentially associated with hippocampal mossy fiber endings in the CA3, whereas synapses in the molecular layers of the CA1 and dentate gyrus double-labeled for liprin-α3. The localization of liprin-α2 and liprin-α3 with excitatory synapses was confirmed in cultured primary hippocampal neurons. Liprin-α4, which poorly co-distributed with presynaptic markers in hippocampus, instead strongly co-localized with VGLUT1 in the cerebellar molecular layer, suggesting its presence in parallel fiber-Purkinje cell synapses. Finally, staining of cultured glial cells indicated that liprin-α1 and liprin-α3 are also associated with astrocytes. We conclude that liprin-α family proteins might perform independent and specialized synaptic and nonsynaptic functions in different regions of the brain.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/β1 and the components of the LL5β-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

Liprins are highly conserved scaffold proteins that regulate cell adhesion, cell migration, and synapse development by binding to diverse target proteins. The molecular basis governing liprin/target interactions is poorly understood. The liprin-α2/CASK complex structure solved here reveals that the three SAM domains of liprin-α form an integrated supramodule that binds to the CASK kinase-like domain. As supported by biochemical and cellular studies, the interaction between liprin-α and CASK is unique to vertebrates, implying that the liprin-α/CASK interaction is likely to regulate higher-order brain functions in mammals. Consistently, we demonstrate that three recently identified X-linked mental retardation mutants of CASK are defective in binding to liprin-α. We also solved the liprin-α/liprin-β SAM domain complex structure, which uncovers the mechanism underlying liprin heterodimerizaion. Finally, formation of the CASK/liprin-α/liprin-β ternary complex suggests that liprins can mediate assembly of target proteins into large protein complexes capable of regulating numerous cellular activities.