NBI Super-Resolution Publications
Selected publications related to our system or services
Normal brain functions depend on the balanced development of excitatory and inhibitory synapses. Our knowledge of the molecular mechanisms underlying inhibitory synapse formation is limited. Neuroligin-2 (NL2), a transmembrane protein at inhibitory postsynaptic sites, is capable of initiating inhibitory synapse formation. In an effort to search for NL2 binding proteins and the downstream mechanisms responsible for inhibitory synapse development, we identify LHFPL4/GARLH4 as a major NL2 binding partner that is specifically enriched at inhibitory postsynaptic sites. LHFPL4/GARLH4 and NL2 regulate the protein levels and synaptic clustering of each other in the cerebellum. Lhfpl4/Garlh4/ mice display profound impairment of inhibitory synapse formation as well as prominent motor behavioral deficits and premature death. Our findings highlight the essential role of LHFPL4/GARLH4 in brain functions by regulating inhibitory synapse formation as a major NL2 binding partner.
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.
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.
Mitotic spindle formation and chromosome segregation require timely separation of the two duplicated centrosomes, and this process is initiated in late G2 by centrosome disjunction. Here we report that GAS2L1, a microtubule- and actin-binding protein, associates with the proximal end of mature centrioles and participates in centriole dynamics and centrosome disjunction. GAS2L1 attaches microtubules and actin to centrosomes, and the loss of GAS2L1 inhibits centrosome disjunction in G2 and centrosome splitting induced by depletion of the centrosome linker rootletin. Conversely, GAS2L1 overexpression induces premature centrosome separation, and this activity requires GAS2L1 association with actin, microtubules, and the microtubule end-binding proteins. The centrosome-splitting effect of GAS2L1 is counterbalanced by rootletin, reflecting the opposing actions of GAS2L1 and the centrosome linker. Our work reveals a GAS2L1-mediated centriole-tethering mechanism of microtubules and actin, which provide the forces required for centrosome dynamics and separation.
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.
Superresolution imaging has revealed subcellular structures and protein interactions in many organisms. However, superresolution microscopy with lateral resolution better than 100 nm has not been achieved in photosynthetic cells due to the interference of a high-autofluorescence background. Here, we developed a photobleaching method to effectively reduce the autofluorescence of cyanobacterial and plant cells. We achieved lateral resolution of ~10 nm with stochastic optical reconstruction microscopy (STORM) in the sphere-shaped cyanobacterium Prochlorococcus and the flowering plant Arabidopsis thaliana. During the cell cycle of Prochlorococcus, we characterized the three-dimensional (3D) organization of the cell division protein FtsZ, which forms a ring structure at the division site and is important for cytokinesis of bacteria and chloroplasts. Although the FtsZ ring assembly process in rod-shaped bacteria has been studied extensively, it has rarely been studied in sphere-shaped bacteria. Similarly to rod-shaped bacteria, our results with Prochlorococcus also showed the assembly of FtsZ clusters into incomplete rings and then complete rings during cell division. Differently from rod-shaped bacteria, the FtsZ ring diameter was not found to decrease during Prochlorococcus cell division. We also discovered a novel double-Z-ring structure, which may be the Z rings of two daughter cells in a predivisional mother cell. Our results showed a quantitative picture of the in vivo Z ring organization of sphere-shaped bacteria.
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.
We report a robust two-color method for super-resolution localization microscopy. Two-dye combination of Alexa647 and Alexa750 in an imaging buffer containing COT and using TCEP as switching regent provides matched and balanced switching characteristics for both dyes, allowing simultaneous capture of both on a single camera. Active sample locking stabilizes sample with 1nm accuracy during imaging. With over 4,000 photons emitted from both dyes, two-color superresolution images with high-quality were obtained in a wide range of samples including cell cultures, tissue sections and yeast cells.
Ataxia-telangiectasia is a multi-systemic 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 six or greater trains. To assess whether the deficit was associated with a pre- or post-synaptic 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 (STORM). Three-dimensional reconstruction revealed that ATM is significantly more closely associated with Piccolo (a pre-synaptic marker) than with Homer1 (a post-synaptic 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.
This paper proposes a cascading algorithm (CSR) based on compressed sensing, which aims to reduce intensive computations in super-resolution imaging of fluorescence microscopy. Performance of existing algorithms such as CVX and L1H drop sharply when applied to obtain finer images with high density molecules. CSR fully exploits the extreme sparsity property of molecules in the compressed sensing model and progressively restricts solution space stage by stage. We perform a comprehensive study of existing algorithms and the proposed algorithm under different resolutions and molecules' densities. Simulation and experimental results confirm the performance advantage of CSR when applied to recover dense molecules.