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.

We report the anchoring of 3D-DNA-cholesterol labeled cages on spherically supported lipid bilayer membranes (SSLBM) formed on silica beads, and their addressability through strand displacement reactions, controlled membrane orientation and templated dimerization. The bilayer-anchored cages can load three different DNA-fluorophores by hybridization to their "top" face (furthest from bilayer) and unload each of them selectively upon addition of a specific input displacement strand. We introduce a method to control strand displacement from their less accessible "bottom" face (closest to the bilayer), by adding cholesterol-substituted displacing strands that insert into the bilayer themselves in order to access the toehold region. The orientation of DNA cages within the bilayer is tunable by positioning multiple cholesterol anchoring units on the opposing two faces of the cage, thereby controlling their accessibility to proteins and enzymes. A population of two distinct DNA cages anchored to the SSLBMs exhibited significant membrane fluidity and have been directed into dimer assemblies on bilayer via input of a complementary linking strand. Displacement experiments performed on these anchored dimers indicate that removal of only one prism's anchoring cholesterol strand was not sufficient to release the dimers from the bilayer; however, removal of both cholesterol anchors from the dimerized prisms via two displacement strands cleanly released the dimers from the bilayer. This methodology allows for the anchoring of DNA cages on supported lipid bilayers, the control of their orientation and accessibility within the bilayer, and the programmable dimerization and selective removal of any of their components. The facile coupling of DNA to other functional materials makes this an attractive method for developing stimuli-responsive protein or nanoparticle arrays, drug releasing biomedical device surfaces and self-healing materials for light harvesting applications, using a highly modular, DNA-economic scaffold.

Spherically supported bilayer lipid membranes (SS-BLMs) exhibiting co-existing membrane microdomains were created on spherical silica substrates. These 5 μm SiO2-core SS-BLMs are shown to interact dynamically when interfaced with living cells in culture, while keeping the membrane structure and lipid domains on the SS-BLM surface intact. Interactions between the SS-BLMs and cellular components are examined via correlating fluorescently labeled co-existing microdomains on the SS-BLMs, their chemical composition and biophysical properties with the consequent organization of cell membrane lipids, proteins, and other cellular components. This approach is demonstrated in a proof-of-concept experiment involving the dynamic organization of cellular cytoskeleton, monitored as a function of the lipid domains of the SS-BLMs. The compositional versatility of SS-BLMs provides a means to address the relationship between the phenomenon of lipid phase separation and the other contributors to cell membrane lateral heterogeneity.

ABSTRACT:The ultrastructural details of presynapses formed between artificial substrates of submicrometer silica beads and hippocampal neurons are visualized via cryoelectron 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.