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Biological and Medical Applications of Materials and Interfaces
A NeuN specific fluorescent probe revealing neuronal nuclei protein and nuclear acids association in living neurons under STED nanoscopy Xiaohe Tian, Tianyan Liu, Bin Fang, Aidong Wang, Mingzhu Zhang, Sajid Hussain, Lei Luo, Ruilong Zhang, Qiong Zhang, Jieying Wu, Giuseppe Battaglia, Lin Li, Zhongping Zhang, and Yupeng Tian ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11102 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018
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A NeuN Specific Fluorescent Probe Revealing Neuronal Nuclei Protein and Nuclear Acids Association in Living Neurons under STED Nanoscopy Xiaohe Tian, ‡abc Tianyan Liu, ‡a Bin Fang, ‡c Aidong Wang, d Mingzhu Zhang, c Sajid Hussain,a Lei Luo,e Ruilong Zhang,c Qiong Zhang,c Jieying Wu,c Giuseppe Battaglia,f Lin Li,g Zhongping Zhang*ch and YupengTian*c a
School of Life Science, Anhui University, Hefei 230601, P. R. China. Institute of Physical Science and Information Technology, Anhui University, Hefei 230601, P. R. China. c Department of Chemistry, Anhui Province Key Laboratory of Chemistry for Inorganic/Organic Hybrid Functionalized Materials, Anhui University, Hefei 230601, P. R. China. d School of Chemistry and Chemical Engineering, Huangshan University, Huangshan 245041, P. R. China e College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, P. R. China f Department of Chemistry, University College London, London, WC1H 0AJ, UK g Key Laboratory of Flexible Electronics & Institute of Advanced Materials, Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University,Nanjing 211816, P.R. China. h CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230000, P. R. China. ‡ These authors contributed equally. b
Abstract: Neuronal nuclei protein (NeuN) is a key RNA-associated protein to guide the transcription of RNA and regulate mRNA splicing in neurons. However, the lack of effective labelling and tracking method has hindered the elucidation of the biological mechanism of NeuN operation in living neurons to understand correlated central nervous system disorders. Here we report a molecular probe that can insert into a neighbouring hydrophobic-hydrophilic region in NeuN, which upon binding becomes capable of emitting light in red region. The NeuN specificity enables the probe imaging neuronal cells in primary brain regions including hippocampus, cerebellum, midbrain and cingulate gyrus. The probes optical properties are such to enable stimulated emission depletion (STED) imaging showing for the first time the 3D structure of RNA tangling into NeuN in a living neuron with tens of nanometer resolution. Keywords: Fluorescent Probe, Neuronal Nuclei Protein, Living Neurons, STED Nanoscopy
1. Introduction Neurons are the main component of the central nervous system (CNS) and they are well organized into complex brain structures including hippocampus, cerebellum and cingulate gyrus, responsible triggering the basic brain functions such as cognitive and emotional through 1 electrical or chemical signals. hence, effective neuron labelling remains a powerful tool to further understand the relevant functions of neurons in different brain domains. Up to date, well-established methods such as 2 Immunofluorescence, lipophilic tracers such as (1,1'Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine 3 4 Perchlorate (DiI) and viral vectors have been widely employed to label neurons for correlated investigation, however these approaches are often time consuming and pose risk to infect primary tissues with poor neuronal specificity. A recent effort was attempted using highthroughput screening to identify neuronal probes but the 5 actual neuronal targeting mechanism remains unclear. We tackled this focusing our attention to a neuronal overexpressed protein, the neuronal nuclei protein (NeuN, 6 also known as RBFOX3). NeuN has been extensively
used as mature neuron marker target (e.g. monoclonal antibodies A60) that specifically expressed only in neuronal nuclei and to some extent the cytoplasm of neuronal cells for most vertebrates,7 and to measure the neuron/glia ratio in brain regions.8 NeuN plays a prominent role in neural tissue development and regulation of adult brain function, while dysfunction or down regulation of NeuN have been associated with several neurological disorders.9 Although NeuN is a nucleic acid binding protein,10 its association with DNA or RNA has never been visualized by imaging. Therefore, staining live neurons in vivo or ex vivo by employing a small molecular probe with high specificity under fluorescent microscopy as well as its extended SR microscopy, could be a valuable approach for researchers.11-12 In this work, we reported a labelling method with a rationally designed molecular probe SL-Neu that specifically binds to NeuN in neurons. The probe fluorescence is only activated upon binding where the protein inhibits its configuration transfer and enhances its emission and two-photon absorption. The high selectivity of SL-Neu towards neuronal tissue and NeuN protein in
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contrast to other parenchymal cells was demonstrated. Due to its superb photo-stability against depletion laser power, we also showed for the first time the nuclear acid association (RNA and DNA) with NeuN protein in living neurons under 3-dimentional stimulated emission depletion nanoscopy (3D-STED). Such functional probe not only offers a powerful tool for specifically isolation neuronal cells, but also provides an insight on visualization and investigation of precise NeuN activities and correlated CNS disease for neurobiology.
2. Materials and Methods 2.1 Preparation of SL1 Using a 100 mL one-necked flask fitted with a stirrer and a condenser, 1.80 g (10 mmol) of 4-((2hydroxyethyl)(methyl)amino) benzaldehyde, 2.15 g (10 mmol) of 3-(4-methylpyridin-1-ium-1-yl) (L) and 30 mL of absolute ethanol were mixed. Five drops of piperidine were added to the mixture and refluxed for 24 h. After cooling, the solution was filtered, and the solid was washed thrice with ether. Red solid product SL1 was collected. Yield: 87.4 %. 1H NMR (400 MHz, d6-DMSO) δ ppm: 8.76 (d, J = 6.6 Hz, 2H), 8.04 (d, J = 6.6 Hz, 2H), 7.91 (d, J = 16.1 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H), 6.94 – 6.68 (m, 2H), 4.55 (t, J = 6.8 Hz, 2H), 3.57 (s, 2H), 3.50 (dd, J = 12.6, 6.6 Hz, 2H), 3.04 (d, J = 5.7 Hz, 4H), 2.43 (t, J = 7.1 Hz, 2H), 2.19 (p, J = 6.9 13 Hz, 2H). C NMR (100 MHz, d6-DMSO) δ ppm: 153.64, 150.27, 143.53, 142.10, 130.38, 122.21, 121.97, 116.59, cm111.47, 58.18, 57.89, 53.77, 47.05, 38.65, 27.11. IR (KBr, 1 ): 3371, 3045, 2905, 2871, 1643, 1589, 152, 1387, 1167, 1040, 967, 837. Anal. Calcd.for C19H24N2O4S: C, 60.62; H, 6.43; N,7.44 %. Found: C, 60.85; H, 6.618; N, 7.455 %. m/z: + 377.1522 ( [M+H] ).
using HyD reflected light detectors (RLDs). Specimen living cells were prepared using similar method as normal confocal microscopy described previously. The STED micrographs were further processed ‘deconvolution wizard’ function using Huygens Professional software (version: 16.05) under authorized license. 2.4 Protein isolation and SDS-PAGE electrophoresis The hippocampus region was isolated from mouse brain and grinded into full homogenate. The isolation of nuclear and cytoplasmic proteins was performed according to the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech, China). The extraction proteins were dissolved in SDS-sample buffer, heated according to the manufacturer’s instruction, and then separated using a 5 % stacking gel and 15 % resolving gel. After electrophoresis, the gels were stained with complex SLNeu (100 µM) for 12 hours. The staining of the gels was measured on the protein- simple Gel Doc imaging system. 2.5 Molecule docking The crystal structure of Neu-Rbfox (ID: 2N82) were retrieved from PDB database, Explicit hydrogen atoms were added, and all water molecules were then deleted. In order to maintain the structural integrity of proteins, there is no treatment of rbfox proteins combined with RNA. To demonstrate the specific recognition of SL1, SL2 and SL3 with respect to Neu (2N82) respectively, docking was carried out using the docking method of ligandfit vina software (version 2016, The Biovia Co.). The parameter was set as default. At the meantime, the binding energy is calculated. For detailed characterizations and experimental information, please refer to Supporting Documents.
2.2 Preparation of SL-Neu Compound SL-Neu was obtained by the same method used for SL1, using 4-(bis(2-hydroxyethyl)amino)benzaldehyde. 1 Yield: 80.8 %. H NMR (400 MHz, d6-DMSO) δ ppm: 8.76 (d, J = 6.3 Hz, 2H), 8.04 (d, J = 6.2 Hz, 2H), 7.90 (d, J = 16.1 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 16.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 2H), 4.83 (s, 2H), 4.54 (t, J = 6.6 Hz, 2H), 3.72 – 3.46 (m, 8H), 2.43 (t, J = 7.0 Hz, 2H), 2.26 – 2.10 (m, 2H). 13 C NMR (100 MHz, d6-DMSO) δ ppm: 153.69, 150.28, 143.57, 142.04, 130.32, 122.24, 122.02, 116.74, 111.60, 58.08, 57.89, 53.07, 47.03, 27.17. IR (KBr, cm-1): 3375 , 3040, 2915, 2877, 1642, 1590, 1521, 1387, 1168, 1038, 965, 840. Anal. Calcd. for C20H26N2O5S: C, 59.09; H, 6.45; N, 6.89 %. Found: C, 56.69; H, 6.613; N, 6.580 %. m/z: 407.1627 ( [M+H] +). 2.3 STED imaging STED nanoscopy experiments was performed under Leica DMi8 confocal microscopy equipped with Leica TCS SP8 STED-ONE unit and the compound was excited under STED laser, the emission signals were collected
3. Results and discussion 3.1 Designs and synthesis The chemical structures of the sulfosalt derivatives with D-π-A (D = donor, A = acceptor) model including SL-Neu, SL-1, SL-2 and SL-3 were shown in Figure 1a. Their detailed synthesis, characterizations and crystal structural information were detailed in the ESI (Scheme S1, Figure S1-S15, Table S1-S3). Their photophysical properties, including nonlinear optical properties were also systemically investigated. (Figure S16-S22, TableS4-S5). It was found that the largest two-photon (2P) action crosssections of probe SL-Neu in PBS buffer solution were located in the range of 840 – 940 nm. The dual-hydroxyl groups in SL-Neu molecule have a tendency to form strong hydrogen-bind (H-bond) with NeuN amino residues. Furthermore, the introduction of sulfo-group favors water solubility (Figure S14) and biocompatibility of the compounds, which in turn allowed avoiding the use of biological unfriendly solvents (e.g. DMSO) and at the
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same time offering the possibility to form H-bind with
NeuN protein, restricting molecular rotation.
Fig. 1 (a) Chemical structure of sulfosalt derivatives including SL-Neu, SL-1, SL-2 and SL-3; Insert table: modelled NeuN binding energy and live neuron specificity. (b) Molecular docking simulated hydrophobic and hydrophilic interaction of between SL-Neu and partial NeuN-Rbfox with indication of protein hydrophobicity; Insert figure: Molecular docking simulated diagram of SL-Neu and whole NeuN-Rbfox. (c) The detailed interaction of SL-Neu and amino acid residue in NeuN protein. GLU164, GLN121, ASP114, ARG109). Notably, the 3.2 Computer simulation interaction with NeuN protein relatively higher binding energy (-70.159 Kcal/mol) and The specific recognition of SL-Neu and other compounds neuronal specificity from SL-Neu molecule (Figure 1a 13 against NeuN-Rbfox protein (PDB: 2N82) was initially insert table and Table S6) might be contributed by the confirmed via molecular docking using the Discover dual-hydroxyl moieties, together with sulfonic acid group Studio ligandfitVina software (version 2016, The Biovia which led the formation of a ‘triangle-like’ H-bind force 14 Co.). The 2N82 PDB structure possess a semi-open within NeuN-Rbfox protein. Subsequently, the restricted cavity consist by a α-helix and a β-sheet, offering the rotation of such D-π-A molecule may also led to possible binding site for SL-Neu molecule (Figure 1b and enhanced intermolecular charge transfer (ICT), similarly Figure S23). In addition, the hydrophobic (Brown) as the influence of its solvent viscosity (Figure S25-S27). hydrophilic (Blue) region in the protein active site (Figure On the other hand, molecules SL1, SL2 and SL3, which 1b) interacts with corresponding hydrophobic/hydrophilic lack the dual-hydroxyl moieties, have no neuronal moieties of SL-Neu. Indeed, the detailed interactions selectivity at later ex vivo experiments. including hydrogen binding and π-sulfur, π-anion (Figure 1c and Figure S24) was clearly presented (MET118,
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Fig. 2 Confocal images of cerebellum, midbrain, cingulate gyrus and hippocampus regions of fresh mouse brain sections incubated with SL-Neu compound (10 µM) for 30 min with high magnification micrographs of the white rectangular regions, with 3-dimension image from selected region from hippocampus. Scale bar = 100 µm.
3.3 The ex vivo assessments of SL-compounds To elucidate the selectivity of these molecules for brain cells, fresh brain sections as well as liver, kidney and heart sections were prepared and incubated using SL-Neu, SL-1, SL-2 and o SL-3 under 37 C and 5 % CO2 for 1 hour. The primary tissue slices were then imaged under confocal laser scanning microscopy without further fixation. Notably, in liver, kidney
and heart sections, the molecules produced negligible signal, while in SL-Neu treated brain sections some regions displayed strong emission (Figure S28). A similar brain uptake pattern was also observed under two-photon confocal microscopy (Figure S29). This reslut was also verified using prefixed tissues that were permeabilized with paraformaldehyde, suggesting that such specificity was present to both living and fixed samples.
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Fig. 3 Colocations studies of SL-Neu compound. (a) Left: Colocations micrographs between β-Tublin and SL-Neu compounds. Right: The magnified micrographs from selected region. (b) Single cell SL-Neu and β-Tublin intensity profile indicated the cellular distribution (c-e) The SL-Neu-stained regions were immunostained with GFAP for astrocytes, AlexaFluor-488 Lectin for Capillary, MAP-2 for neuronal fibrils, respectively. (f) SL-Neu-stained regions of mouse brain and spinal cord were immunostained with NeuN antibody. (g) SDS-PAGE analysis of protein from mouse nerve cell extracted from fresh brain hippocampus upon staining with Coomassie brilliant blue R-250 and SL-Neu. Coomassie brilliant blue R-250 staining (left). SLNeu molecules staining under ultraviolet transillumination. Scale bar = 20 µm.
3.4 Subcellular neuronal distribution of SL-Neu To further examine such specificity, brain sections with different neurons rich regions including cerebellum, midbrain, cingulate gyrus and hippocampus were evaluated. As Figure 2 and its enlarged micrographs showed, distinct signal was detected and these might highly correspondent to neuronal cells. Its neuronal specificity was confirmed when the brain cells were stained with the classic DNA dye DAPI (4’, 6diamidino-2-phenylindole), only cells within the hippocampus region displayed positive signal (Figure S30). In contrast, other cells (non-neuronal cells indicated by white arrows) showed negligible signals, suggesting the reduced uptake of SL-Neu molecules. To verify the probe’s intracellular distribution, β-Tublin 15 antibody was used to label microtubules-rich neuron cells after incubating with SL-Neu molecule. As displayed in Figure 3a in the magnified micrograph, β-Tublin demonstrated elongated neuronal fibril morphology and its signal were excluded from nuclear region. In contrast, SL-Neu displayed
much less cytosolic distribution that partially overlapped with β-Tublin, while it showed intense nuclear internalization. The uptake pattern was further confirmed as intensity plot profile as indicated in Figure 3b. Such cytosolic and nuclear observation is not surprising, since NeuN protein could be both expressed only in neuronal nuclei and to some extent the cytoplasm. This further supports that SL-Neu compound was NeuN specific. 3.5 Confirmation of SL-Neu specificity against NeuN protein In order to confirm the selectivity of SL-Neu against neuronal NeuN protein, a series of immunofluorescence experiments were performed to label brain parenchymal cells and another neuronal marker. As one of the most representative cell population in the brain, astrocytes can be effectively stained using glial fibrillary acidic protein (GFAP) antibody. Figure 3c demonstrated a few astrocytes closely associated with SLNeu-positive cells from hippocampus region but without overlapping, as indicated by co-localization coefficient analysis via Pearson’s Rr (right bar-chart). Brian
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Fig. 4 (a) Confocal images of live neuron treated with SL-Neu and NucRed (DNA), Syto-9(RNA). (b) The corresponding STED images showing the combination of SL-Neu and DNA, RNA respectively under high resolution. (c) and (d) The fluorescent intensity profile of the lines in chromatin region. (e) and (f) 3-dimension STED micrographs indicating the association of SL-Neu and RNA.
endothelial cell is the main component of blood brain barrier that exist within brain parenchyma, responsible for substances exchange between central nervous system and the rest of body. As Figure 3d shown SL-Neu positive cells display no co-localization with blood vessels, again suggesting a very high specificity of SL-Neu molecule towards a specific cell type, which might correspond to neurons. Subsequently, MAP2 (microtubule-associated protein 2, a common marker of neuronal axons) was used to specifically label neuronal fibrils, and showed little overlap with SL-Neu signal. Based on the aforementioned results such as strong intracellular fluorescence of SL-Neu in neurons rich regions, high intensity in nuclear region of β-tubulin rich neuronal cells, non-specific interaction for non-neuronal cells and neuronal MAP2, co-staining with NeuN antibody was eventually performed. Here brain and spinal cord of mouse were harvested, their slices were prepared and incubated with SLNeu followed with standard immunofluorescence protocol. Figure 3f clearly presents high degree of co-localization in both brain and spinal cord sections (Figure S31-S32). Generally, NeuN reactivity is mainly nuclear, whereas it may also be found in the cytoplasm of numerous neuronal cell types. Nonetheless, these immunofluorescence experiments strongly suggested SL-Neu molecules could effectively and specifically penetrate into live neuronal cells and internalize
with neuronal nuclei protein, in a good agreement with our initial design purpose and computation modelling results. To further confirm whether the staining of SL-Neu was indeed associated with the neuronal nuclei (NeuN) protein, nuclear and cytoplasmic proteins of mouse nerve cell were isolated from the hippocampus region, and protein electrophoresis was carried out. After electrophoresis, the gels were stained with SL-Neu (10 µM) for 12 h, and Coomassie brilliant blue was used as a reference. As shown in Figure 3g, SL-Neu molecules manifested the staining specificity of NeuN protein distributed in nucleus and cytoplasm by detecting two major bands (nuclear) and two weak bands (cytoplasm), highly correspondent to NeuN/Rbfox46- and 48-kDa subtypes. This result was in good agreement with imaging result, which showed SL-Neu signal and the NeuN protein signal with a considerably high overlay rate, again strongly suggesting SLNeu was a NeuN specific fluorescent probe in either live or fixed neuronal cells. 3.6 demonstrate nuclear acid and NeuN association using SLNeu under STED nanoscopy The utilization of SL-Neu molecules under stimulated emission depletion (STED) nanoscopy to reveal the association between NeuN protein and nuclear acid was also performed. Although some literature data suggests that NeuN is a pre-mRNA alternative splicing regulator and binds to 10 RNA, the correlation between NeuN and DNA or RNA in
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living neurons under super-resolution nanoscopy has never been demonstrated elsewhere, due to the lack of effective probes. Since STED technique require intense normal confocal laser along with a depletion laser, photo-stability is 16 critical, hence SL-Neu photo-resistance under STED condition was evaluated. Live brain section incubated with SLNeu and exposed under STED laser over 400-times continued scans. Intensity profile suggested such compound displayed super- stability under both confocal and STED condition (Figure S33). Subsequently, in order to display the ultra-detailed correlations between SL-Neu and nuclear acid, we performed co-localization experiments using commercial dyes NucRed and Syto9 for DNA and RNA, respectively. Conventional live cell imaging was firstly obtained, suggesting the compatibility of SL-Neu molecules with those nuclear acids marker under confocal microscopy (Figure 4a and Figure S34). Due to the remarkable photo-stability and red emission of SL-Neu molecules, STED super resolution micrographs were captured. Figure 4b indicated the nuclear NeuN protein was successfully marked with high signal-to-noise contrast. Neighboring chromatins structures were also revealed and the corresponding x-y plot profile indicating a ~55 nm distance between NeuN and DNA. In contrast, RNA showed much more overlapping signal with less than ~ 20 nm distance was verified. Indeed, 3D super resolution single nucleolar imaging, with 85 Z-sections (Figure 4e), was successfully obtained and indicated the actual binding details of NeuN protein within the RNA entangled nucleoli. In contrast, DNA displayed much less interaction with SL-Neu signal (Figure S35). Although previous studies suggested NeuN protein could internalize with DNA in vitro, our observation using SL-Neu as a probe might strengthen that NeuN protein is a RNA associate protein in living neurons.
4. Conclusions In summary, we have rationally designed a small fluorescent probe SL-Neu that can selectively target live neuron NeuN protein. SL-Neu live neuron labeling coupled with NeuN binding properties rendered further observation of NeuN and nuclear acid association using super-resolution technique under STED nanoscopy. This innovative compound serves as an imaging tool for living neurons as well as to shed light to correlated cellular mechanisms and ultimately NeuN associated CNS diseases.
Supporting Information Detailed synthesis, characterization, tissue labelling and super-resolution work can be found in supporting files, is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Authors *E-mail:
[email protected] (Z.Z)
*E-mail:
[email protected] (Y.T.). Conflicts of interest There are no conflicts to declare.
Acknowledgements This work was supported by a grant for the National Natural Science Foundation of China (21602003, 51432001, 51672002, 51772002, 81503009 and 21501001), Anhui University Doctor Startup Fund (J01001962). X.T thank Anhui Provincial Natural Science Foundation of China (1708085MC68), Anhui Provincial Returnees Innovation and Entrepreneurship key support program, Open fund for Discipline Construction， Institute of Physical Science and Information Technology, Anhui University. Giuseppe Battaglia thanks the Anhui Distinguished Foreign Expert program.
Notes and references 1. Nicholls, J. G.; Martin, A. R.; Wallace, B. G.; Fuchs, P. A., From Neuron to Brain. Sinauer Associates Sunderland, MA: 2001; Vol. 271. 2. Kádár, A.; Wittmann, G.; Liposits, Z.; Fekete, C., Improved Method for Combination of Immunocytochemistry and Nissl Staining. Journal of neuroscience methods 2009, 184, 115-118. 3. Schepartz, A.; Thompson, A. D.; Omar, M. H.; Rivera-Molina, F.; Xi, Z.; Koleske, A. J.; Toomre, D. K., Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR. Angewandte Chemie International Edition 2017. 56,10408-10412. 4. Foust, K. D.; Nurre, E.; Montgomery, C. L.; Hernandez, A.; Chan, C. M.; Kaspar, B. K., Intravascular AAV9 Preferentially Targets Neonatal Neurons and Adult Astrocytes. Nature biotechnology 2009, 27, 59-65. 5. Er, J. C.; Leong, C.; Teoh, C. L.; Yuan, Q.; Merchant, P.; Dunn, M.; Sulzer, D.; Sames, D.; Bhinge, A.; Kim, D., NeuO: A Fluorescent Chemical Probe for Live Neuron Labeling. Angewandte Chemie International Edition 2015, 54, 2442-2446. 6. Kim, K. K.; Adelstein, R. S.; Kawamoto, S., Identification of neuronal nuclei (NeuN) as Fox-3, A New Member of the Fox-1 Gene Family of Splicing Factors. Journal of Biological Chemistry 2009, 284, 3105231061. 7. Weyer, A.; Schilling, K., Developmental and Cell Type-specific Expression of the Neuronal Marker NeuN in the Murine Cerebellum. Journal of neuroscience research 2003, 73, 400-409. 8. Herculano-Houzel, S.; Lent, R., Isotropic Fractionator: A Simple, Rapid Method for the Quantification of Total Cell and Neuron Numbers in the
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Brain. Journal of Neuroscience 2005, 25, 2518-2521. 9. Duan, W.; Zhang, Y.-P.; Hou, Z.; Huang, C.; Zhu, H.; Zhang, C.-Q.; Yin, Q., Novel insights into NeuN: From Neuronal Marker to Splicing Regulator. Molecular neurobiology 2016, 53, 1637-1647. 10. Dent, M. A.; Segura-Anaya, E.; Alva-Medina, J.; Aranda-Anzaldo, A., NeuN/Fox-3 is an Intrinsic Component of the Neuronal Nuclear Matrix. FEBS letters 2010, 584, 2767-2771. 11. Kim, H. M.; Cho, B. R., Small-molecule Two-photon Probes for Bioimaging Applications. Chemical reviews 2015, 115, 5014-5055. 12. Vicidomini, G.; Bianchini, P.; Diaspro, A., STED Super-resolved Microscopy. Nature methods 2018. 15,173-182 13. Chen, Y.; Zubovic, L.; Yang, F.; Godin, K.; Pavelitz, T.; Castellanos, J.; Macchi, P.; Varani, G., Rbfox Proteins Regulate MicroRNA Biogenesis by SequenceSpecific Binding to Their Precursors and Target Downstream Dicer. Nucleic acids research 2016, 44, 4381-4395. 14. Biovia, D. S., Discovery Studio Modeling Environment, Release 2017. 2016. 15. Kapitein, L. C.; Hoogenraad, C. C., Building the Neuronal Microtubule Cytoskeleton. Neuron 2015, 87, 492-506. 16. Schermelleh, L.; Heintzmann, R.; Leonhardt, H., A Guide to Super-resolution Fluorescence Microscopy. The Journal of cell biology 2010, 190, 165-175.
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