ATM and ATR play complementary roles in the behavior of excitatory and inhibitory vesicle populations.

ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) are large PI3 kinases whose human mutations result in complex syndromes that include a compromised DNA damage response (DDR) and prominent nervous system phenotypes. Both proteins are nuclear-localized in keeping with their DDR functions, yet both are also found in cytoplasm, including on neuronal synaptic vesicles. In ATM- or ATR-deficient neurons, spontaneous vesicle release is reduced, but a drop in ATM or ATR level also slows FM4-64 dye uptake. In keeping with this, both proteins bind to AP-2 complex components as well as to clathrin, suggesting roles in endocytosis and vesicle recycling. The two proteins play complementary roles in the DDR; ATM is engaged in the repair of double-strand breaks, while ATR deals mainly with single-strand damage. Unexpectedly, this complementarity extends to these proteins' synaptic function as well. Superresolution microscopy and coimmunoprecipitation reveal that ATM associates exclusively with excitatory (VGLUT1+) vesicles, while ATR associates only with inhibitory (VGAT+) vesicles. The levels of ATM and ATR respond to each other; when ATM is deficient, ATR levels rise, and vice versa. Finally, blocking NMDA, but not GABA, receptors causes ATM levels to rise while ATR levels respond to GABA, but not NMDA, receptor blockade. Taken together, our data suggest that ATM and ATR are part of the cellular "infrastructure" that maintains the excitatory/inhibitory balance of the nervous system. This idea has important implications for the human diseases resulting from their genetic deficiency.

Cdk5-dependent phosphorylation of liprinα1 mediates neuronal activity-dependent synapse development.

The experience-dependent modulation of brain circuitry depends on dynamic changes in synaptic connections that are guided by neuronal activity. In particular, postsynaptic maturation requires changes in dendritic spine morphology, the targeting of postsynaptic proteins, and the insertion of synaptic neurotransmitter receptors. Thus, it is critical to understand how neuronal activity controls postsynaptic maturation. Here we report that the scaffold protein liprinα1 and its phosphorylation by cyclin-dependent kinase 5 (Cdk5) are critical for the maturation of excitatory synapses through regulation of the synaptic localization of the major postsynaptic organizer postsynaptic density (PSD)-95. Whereas Cdk5 phosphorylates liprinα1 at Thr701, this phosphorylation decreases in neurons in response to neuronal activity. Blockade of liprinα1 phosphorylation enhances the structural and functional maturation of excitatory synapses. Nanoscale superresolution imaging reveals that inhibition of liprinα1 phosphorylation increases the colocalization of liprinα1 with PSD-95. Furthermore, disruption of liprinα1 phosphorylation by a small interfering peptide, siLIP, promotes the synaptic localization of PSD-95 and enhances synaptic strength in vivo. Our findings collectively demonstrate that the Cdk5-dependent phosphorylation of liprinα1 is important for the postsynaptic organization during activity-dependent synapse development.

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation.

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.

ATM protein is located on presynaptic vesicles and its deficit leads to failures in synaptic plasticity.

Ataxia telangiectasia is a multisystemic disorder that includes a devastating neurodegeneration phenotype. The ATM (ataxia-telangiectasia mutated) protein is well-known for its role in the DNA damage response, yet ATM is also found in association with cytoplasmic vesicular structures: endosomes and lysosomes, as well as neuronal synaptic vesicles. In keeping with this latter association, electrical stimulation of the Schaffer collateral pathway in hippocampal slices from ATM-deficient mice does not elicit normal long-term potentiation (LTP). The current study was undertaken to assess the nature of this deficit. Theta burst-induced LTP was reduced in Atm(-/-) animals, with the reduction most pronounced at burst stimuli that included 6 or greater trains. To assess whether the deficit was associated with a pre- or postsynaptic failure, we analyzed paired-pulse facilitation and found that it too was significantly reduced in Atm(-/-) mice. This indicates a deficit in presynaptic function. As further evidence that these synaptic effects of ATM deficiency were presynaptic, we used stochastic optical reconstruction microscopy. Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a presynaptic marker) than with Homer1 (a postsynaptic marker). These results underline how, in addition to its nuclear functions, ATM plays an important functional role in the neuronal synapse where it participates in the regulation of presynaptic vesicle physiology.

Vertically aligned ZnO/amorphous-Si core-shell heterostructured nanowire arrays.

We report the synthesis of vertically aligned ZnO/a-Si core-shell nanowire arrays (ZnO nanowires coated with amorphous silicon) through chemical vapor deposition. The core-shell heterostructured nanowires possessed uniform morphology and the thickness of the amorphous silicon shells could be controlled easily by tuning the deposition duration and temperature. The core-shell heterostructured nanowires exhibited enhanced antireflection and absorption performance as well as tunable PL properties. Because the individual ZnO/a-Si nanowires showed p-type characteristics and the ZnO cores were n-type semiconductors, the core-shell nanowires formed p-n junctions naturally.

Visualization of Protein Sorting at the Trans-Golgi Network and Endosomes Through Super-Resolution Imaging.

The trans-Golgi network (TGN) and endosomes are essential protein sorting stations in the secretory transport pathway. Protein sorting is fundamentally a process of spatial segregation, but the spatial relationships among the proteins that constitute the sorting machinery have not been systematically analyzed at high resolution in mammalian cells. Here, using two-color STORM imaging, we show that the TGN/endosome-localized cargo adaptors, AP-1, GGA2 and epsinR, form elongated structures of over 250 nm in length at the juxta-nuclear Golgi area. Many of these structures are associated with clathrin. We found that AP-1 is spatially segregated from AP-3 and GGA2, whereas a fraction of AP-1 and GGA2 punctae are associated with epsinR. Moreover, we observed that the planar cell polarity cargo proteins, Vangl2 and Frizzled6 associate with different cargo adaptors-AP-1 and GGA2 or epsinR, respectively-when exiting the TGN. Knockdown analysis confirms the functional significance of this segregation. Our data indicates that TGN/endosome-localized cargo adaptors have distinct spatial relationships. The spatially segregated cargo adaptors GGA2 and AP-1 regulate sorting of Frizzled6 and Vangl2, respectively and spatially associated cargo adaptors can cooperatively regulate a specific sorting process.

A Mitochondrion-Specific Photoactivatable Fluorescence Turn-On AIE-Based Bioprobe for Localization Super-Resolution Microscope.

A novel mitochondrion-specific photo-activatable fluorescence turn-on bioprobe, named as o-TPE-ON+, is designed and readily prepared, operating through a new photoactivatable mechanism of photocyclodehydrogenation. This bioprobe exhibits unique photoactivation behavior in cells, and is applied to super-resolution imaging of mitochondrion and its dynamic investigation in both fixed and live cells under physiological conditions without any external additives.

Mechanosensitive gating of CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion and intracellular ligand-gated channel associated with cystic fibrosis, a lethal genetic disorder common among Caucasians. Here we show that CFTR is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular and tissue levels. Stretch increased the product of the number of channels present and probability of being open (NPo), and also increased the unitary conductance of CFTR in cell-attached membrane patches. CFTR stretch-mediated activation appears to be an intrinsic property independent of cytosolic factors and kinase signalling. CFTR stretch-mediated activation resulted in chloride transport in Calu-3 human airway epithelial cells and mouse intestinal tissues. Our study has revealed an unexpected function of CFTR in mechanosensing, in addition to its roles as a ligand-gated anion channel and a regulator of other membrane transporters, demonstrating for the first time a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation in vivo.

High-quality ZnO nanowire arrays directly fabricated from photoresists.

We report a simple and effective method for fabricating and patterning high-quality ZnO nanowire arrays using carbonized photoresists to control the nucleation site, density, and growth direction of the nanowires. The ZnO nanowires fabricated using this method show excellent alignment, crystal quality, and optical properties that are independent of the substrates. The carbonized photoresists provide perfect nucleation sites for the growth of aligned ZnO nanowires and they also perfectly connect to the nanowires to form ideal electrodes that can be used in many applications of ZnO nanomaterials.