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Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryo-electron microscopy (cryo-EM). The silica beads are derivatized by poly-d-lysine or lipid bilayers. Molecular features known to exist at presynapses are clearly present at these artificial synapses, as visualized by cryo-EM. Key synaptic features such as the membrane contact area at synaptic junctions, the presynaptic bouton containing presynaptic vesicles, as well as microtubular structures can be identified. This is the first report of the direct, label-free observation of ultrastructural details of artificial synapses.

The versatility of perfluorophenyl azide (PFPA) derivatives makes them useful for attaching a wide variety of biomolecules and polymers to surfaces. Herein, a single molecule force spectroscopy (SMFS) study of the concanavalin A/mannose interaction was carried out using PFPA immobilization chemistry. SMFS of the concanavalin A/mannose interaction yielded an average unbinding force of 70-80 pN for loading rates between 8000 and 40,000 pN/s for mannose surfaces on aminated glass, and an unbinding force of 57 ± 20 pN at 6960 pN/s for mannose surfaces on gold-coated glass. Dynamic force spectroscopy was used to determine the dissociation rate constant, k(off), for this interaction to be 0.16 s(-1).