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author:("Yang, feidong")
1.  High-resolution three-dimensional mapping of mRNA export through the nuclear pore 
Nature communications  2013;4:2414.
The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the center of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.
doi:10.1038/ncomms3414
PMCID: PMC3800679  PMID: 24008311
2.  Role of Molecular Charge in Nucleocytoplasmic Transport 
PLoS ONE  2014;9(2):e88792.
Transport of genetic materials and proteins between the nucleus and cytoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). A selective barrier formed by phenylalanine-glycine (FG) nucleoporins (Nups) with net positive charges in the NPC allows for passive diffusion of signal-independent small molecules and transport-receptor facilitated translocation of signal-dependent cargo molecules. Recently, negative surface charge was postulated to be another essential criterion for selective passage through the NPC. However, the charge-driven mechanism in determining the transport kinetics and spatial transport route for either passive diffusion or facilitated translocation remains obscure. Here we employed high-speed single-molecule fluorescence microscopy with an unprecedented spatiotemporal resolution of 9 nm and 400 µs to uncover these mechanistic fundamentals for nuclear transport of charged substrates through native NPCs. We found that electrostatic interaction between negative surface charges on transiting molecules and the positively charged FG Nups, although enhancing their probability of binding to the NPC, never plays a dominant role in determining their nuclear transport mode or spatial transport route. A 3D reconstruction of transport routes revealed that small signal-dependent endogenous cargo protein constructs with high positive surface charges that are destined to the nucleus, rather than repelled from the NPC as suggested in previous models, passively diffused through an axial central channel of the NPC in the absence of transport receptors. Finally, we postulated a comprehensive map of interactions between transiting molecules and FG Nups during nucleocytoplasmic transport by combining the effects of molecular size, signal and surface charge.
doi:10.1371/journal.pone.0088792
PMCID: PMC3928296  PMID: 24558427
3.  Enhancement of Cerenkov Luminescence Imaging by Dual Excitation of Er3+, Yb3+-Doped Rare-Earth Microparticles 
PLoS ONE  2013;8(10):e77926.
Cerenkov luminescence imaging (CLI) has been successfully utilized in various fields of preclinical studies; however, CLI is challenging due to its weak luminescent intensity and insufficient penetration capability. Here, we report the design and synthesis of a type of rare-earth microparticles (REMPs), which can be dually excited by Cerenkov luminescence (CL) resulting from the decay of radionuclides to enhance CLI in terms of intensity and penetration. Methods: Yb3+- and Er3+- codoped hexagonal NaYF4 hollow microtubes were synthesized via a hydrothermal route. The phase, morphology, and emission spectrum were confirmed for these REMPs by power X-ray diffraction (XRD), scanning electron microscopy (SEM), and spectrophotometry, respectively. A commercial CCD camera equipped with a series of optical filters was employed to quantify the intensity and spectrum of CLI from radionuclides. The enhancement of penetration was investigated by imaging studies of nylon phantoms and nude mouse pseudotumor models. Results: the REMPs could be dually excited by CL at the wavelengths of 520 and 980 nm, and the emission peaks overlaid at 660 nm. This strategy approximately doubled the overall detectable intensity of CLI and extended its maximum penetration in nylon phantoms from 5 to 15 mm. The penetration study in living animals yielded similar results. Conclusions: this study demonstrated that CL can dually excite REMPs and that the overlaid emissions in the range of 660 nm could significantly enhance the penetration and intensity of CL. The proposed enhanced CLI strategy may have promising applications in the future.
doi:10.1371/journal.pone.0077926
PMCID: PMC3808356  PMID: 24205030
4.  AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury 
PLoS ONE  2013;8(7):e69910.
Aims
Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.
Methods
Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by 18F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.
Results
IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).
Conclusions
The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.
doi:10.1371/journal.pone.0069910
PMCID: PMC3724784  PMID: 23922853
5.  Noninvasive Visualization of MicroRNA-16 in the Chemoresistance of Gastric Cancer Using a Dual Reporter Gene Imaging System 
PLoS ONE  2013;8(4):e61792.
MicroRNAs (miRNAs) have been implicated to play a central role in the development of drug resistance in a variety of malignancies. However, many studies were conducted at the in vitro level and could not provide the in vivo information on the functions of miRNAs in the anticancer drug resistance. Here, we introduced a dual reporter gene imaging system for noninvasively monitoring the kinetic expression of miRNA-16 during chemoresistance in gastric cancer both in vitro and in vivo. Human sodium iodide symporter (hNIS) and firefly luciferase (Fluc) genes were linked to form hNIS/Fluc double fusion reporter gene and then generate human gastric cancer cell line NF-3xmir16 and its multidrug resistance cell line NF-3xmir16/VCR. Radioiodide uptake and Fluc luminescence signals in vitro correlated well with viable cell numbers. The luciferase activities and radioiodide uptake in NF-3xmir16 cells were remarkably repressed by exogenous or endogenous miRNA-16. The NF-3xmir16/VCR cells showed a significant increase of 131I uptake and luminescence intensity compared to NF-3xmir16 cells. The radioactivity from in vivo 99mTc-pertechnetate imaging and the intensity from bioluminescence imaging were also increased in NF-3xmir16/VCR compared with that in NF-3xmir16 tumor xenografts. Furthermore, using this reporter gene system, we found that etoposide (VP-16) and 5-fluorouracil (5-FU) activated miRNA-16 expression in vitro and in vivo, and the upregulation of miRNA-16 is p38MAPK dependent but NF-κB independent. This dual imaging reporter gene may be served as a novel tool for in vivo imaging of microRNAs in the chemoresistance of cancers, as well as for early detection and diagnosis in clinic.
doi:10.1371/journal.pone.0061792
PMCID: PMC3629136  PMID: 23613938
6.  Injectable Perlecan Domain 1-Hyaluronan Microgels Potentiate the Cartilage Repair Effect of BMP2 in a Murine Model of Early Osteoarthritis 
The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2 free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans, and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2 treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.
doi:10.1088/1748-6041/7/2/024109
PMCID: PMC3367563  PMID: 22455987
Perlecan; Hyaluronic Acid; Heparan Sulfate; Osteoarthritis; Cartilage Repair; Bone Morphogenetic Protein
7.  Efficient multicistronic co-expression of hNIS and hTPO in prostate cancer cells for nonthyroidal tumor radioiodine therapy 
Radioiodine therapy has proven to be a safe and effective approach in the treatment of differentiated thyroid cancer. Similar treatment strategies have been exploited in nonthyroidal malignancies by transfecting hNIS gene into tumor cells or xenografts. However, rapid radioiodine efflux is often observed after radioiodine uptake, limiting the overall antitumor effects. In this study, we aimed at constructing multicistronic co-expression of hNIS and hTPO genes in tumor cells to enhance the radioiodine uptake and prolong the radioiodine retention. Driven by the cytomegalovirus promoter, hNIS and hTPO were simultaneously inserted into the expression cassette of adenoviral vector. An Ad5 viral vector (Ad-CMV-hTPO-T2A-hNIS) was assembled as a gene therapy vehicle by Gateway technology and 2A method. The co-expression of hNIS and hTPO genes was confirmed by a double-label immunofluorescence assay. The radioiodine (125I) uptake and efflux effects induced by co-expression of hNIS and hTPO genes were determined in transfected and non-transfected PC-3 cells. Significantly higher uptake (6.58 ± 0.56 fold, at 1 h post-incubation) and prolonged retention (5.47 ± 0.36 fold, at 1 h of cell efflux) of radioiodine (125I) were observed in hNIS and hTPO co-expressed PC-3 cells as compared to non-transfected PC-3 cells. We concluded that the new virus vector displayed favorable radioiodine uptake and retention properties in hNIS-hTPO transfected PC-3 cells. Our study will provide valuable information on improving the efficacy of hNIS-hTPO co-mediated radioiodine gene therapy.
PMCID: PMC3484425  PMID: 23145364
Gene therapy; prostate cance; hNIS; hTPO; gateway cloning system
8.  Single-molecule studies of nucleocytoplasmic transport: from one dimension to three dimensions 
Integrative Biology  2011;4(1):10-21.
In eukaryotic cells, the bidirectional trafficking of proteins and genetic materials across the double-membrane nuclear envelope is mediated by nuclear pore complexes (NPCs). A highly selective barrier formed by the phenylalanine–glycine (FG)-nucleoporin (Nup) in the NPC allows for two transport modes: passive diffusion and transport receptor-facilitated translocation. Strict regulation of nucleocytoplasmic transport is crucial for cell survival, differentiation, growth and other essential activities. However, due to the limited knowledge of the native configuration of the FG-Nup barrier and the interactions between the transiting molecules and the barrier in the NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics of individual fluorescent molecules through the NPC and to map the interactions between transiting molecules and the FG-Nup barrier. Important characteristics of nucleocytoplasmic transport, such as transport time, transport efficiency and spatial distribution of single transiting molecules in the NPC, have been obtained that could not be measured by either ensemble average methods or conventional electron microscopy. In this critical review, we discuss the development of various single-molecule techniques and their application to nucleocytoplasmic transport in vitro and in vivo. In particular, we highlight a recent advance from one-dimensional to three-dimensional single-molecule characterization of transport through the NPC and present a comprehensive understanding of the nucleocytoplasmic transport mechanism obtained by this new technical development (105 references).
doi:10.1039/c1ib00041a
PMCID: PMC3387808  PMID: 22020388
9.  Calcium regulation of nucleocytoplasmic transport 
Protein & cell  2011;2(4):291-302.
Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calciumregulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule superresolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.
doi:10.1007/s13238-011-1038-x
PMCID: PMC3387809  PMID: 21528351
nuclear envelope; nuclear pore complex; nucleocytoplasmic transport; calcium stores; single-molecule fluorescence microscopy
10.  Three-dimensional Noninvasive Monitoring Iodine-131 Uptake in the Thyroid Using a Modified Cerenkov Luminescence Tomography Approach 
PLoS ONE  2012;7(5):e37623.
Background
Cerenkov luminescence tomography (CLT) provides the three-dimensional (3D) radiopharmaceutical biodistribution in small living animals, which is vital to biomedical imaging. However, existing single-spectral and multispectral methods are not very efficient and effective at reconstructing the distribution of the radionuclide tracer. In this paper, we present a semi-quantitative Cerenkov radiation spectral characteristic-based source reconstruction method named the hybrid spectral CLT, to efficiently reconstruct the radionuclide tracer with both encouraging reconstruction results and less acquisition and image reconstruction time.
Methodology/Principal Findings
We constructed the implantation mouse model implanted with a 400 µCi Na131I radioactive source and the physiological mouse model received an intravenous tail injection of 400 µCi radiopharmaceutical Iodine-131 (I-131) to validate the performance of the hybrid spectral CLT and compared the reconstruction results, acquisition, and image reconstruction time with that of single-spectral and multispectral CLT. Furthermore, we performed 3D noninvasive monitoring of I-131 uptake in the thyroid and quantified I-131 uptake in vivo using hybrid spectral CLT. Results showed that the reconstruction based on the hybrid spectral CLT was more accurate in localization and quantification than using single-spectral CLT, and was more efficient in the in vivo experiment compared with multispectral CLT. Additionally, 3D visualization of longitudinal observations suggested that the reconstructed energy of I-131 uptake in the thyroid increased with acquisition time and there was a robust correlation between the reconstructed energy versus the gamma ray counts of I-131 (). The ex vivo biodistribution experiment further confirmed the I-131 uptake in the thyroid for hybrid spectral CLT.
Conclusions/Significance
Results indicated that hybrid spectral CLT could be potentially used for thyroid imaging to evaluate its function and monitor its treatment for thyroid cancer.
doi:10.1371/journal.pone.0037623
PMCID: PMC3358266  PMID: 22629431
11.  Efficacy of 99mTc-EDDA/HYNIC-TOC SPECT/CT scintigraphy in Graves’ ophthalmopathy 
The aim of this study was to investigate the predictive role of the orbital somatostatin receptor scintigraphy with 99mTc-EDDA/HYNIC-TOC (99mTc-TOC) to detect clinical stage of Graves’ ophthalmopathy and the response to corticosteroid therapy. The subjects of the experiment were 46 patients with Graves’ ophthalmopathy (GO) and four volunteers without eye disease or GO as the normal group (NG). Single photon emission computed tomography (SPECT), computed tomography (CT) and the left and right lateral position planar imaging of the heads of the all subjects were obtained 4 h after the intravenous injection of 555 MBq of 99mTc-TOC. The 99mTc-TOC SPECT/CT was repeated 3 months later. 35 (35/46) patients were received corticosteroid therapy (prednisolone, 10 mg po tid ) for 3 months, however, the other 11 patients as control groups did not receive any treatment. The treatment effect was evaluated both by the orbital 99mTc-TOC uptake and NOSPECS. A significant decrease in the O/OC ratio was observed in 22 GO patients between pre- and post-treatment (1.64 ± 0.13 vs. 1.21 ± 0.09, P < 0.05). There were neither significant difference of the O/OC ratio in 13 GO patients between pre- and post-treatment periods, nor significant difference in the 9 (9/11) patients before and after three months. Orbital 99mTc-TOC scintigraphy is a feasible technique to estimate the Graves’ ophthalmopathy activity and predict the response to subsequent corticosteroid therapy in GO patients. The technique could be a useful tool for physicians not familiar with CAS determination.
PMCID: PMC3477727  PMID: 23133815
Graves’ ophthalmopathy; single photon emission computed tomography (SPECT); somatostatin receptor; 99mTc-TOC
12.  ‘Natively unfolded’ nucleoporins in nucleocytoplasmic transport 
Nucleus  2011;2(1):10-16.
The nuclear pore complex (NPC) acts as a selective gate that mediates the bidirectional transport of macromolecules between the cytoplasm and the nucleus of eukaryotic cells. ‘Natively unfolded’ nucleoporins (Nups) with domains rich in phenylalanine-glycine (FG) repeats form the selective permeability barrier and provide binding sites for mobile transport receptors in the NPC. Understanding the structure and function of the FG-Nups barrier under real-time trafficking conditions is still a formidable challenge due to the dynamic nature of a channeled membranous environment. Recently, we have shown that three-dimensional (3D) density maps of transient interactions between the FG-Nups barrier and a cargo-free or a cargo-bound transport receptor in native NPCs can be obtained by an advanced single-molecule fluorescence microscopy approach. Moreover, we found that these interaction sites are spatially clustered into distinct groups in the periphery around a central axial channel with a diameter of approximately 10–20 nm in the NPC. The 3D distribution of interaction sites may indicate some native properties of the FG-Nups barrier. Here we speculate that the selective permeability barrier in the NPC could be formed by clustered FG-Nups.
doi:10.4161/nucl.2.1.13818
PMCID: PMC3104804  PMID: 21647294
nucleocytoplasmic transport; single-molecule tracking; three-dimensional distribution; real-time trafficking; super-resolution microscopy; single-molecule fluorescence
13.  Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release 
Biomaterials  2009;30(36):6964-6975.
We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein 2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP-P1) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP-P1 exhibited significantly (p<0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP-P1, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP-P1 exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP-P1 stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP-P1 to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.
doi:10.1016/j.biomaterials.2009.09.009
PMCID: PMC2783995  PMID: 19775743
perlecan domain I; hyaluronic acid; hydrogel particles; controlled release; bone morphogenetic protein-2; chondrogenic differentiation; mesenchymal stem cells
14.  Modulation of microRNA processing and expression through RNA editing by ADAR deaminases 
Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri–miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri–miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.
doi:10.1038/nsmb1041
PMCID: PMC2950615  PMID: 16369484
15.  ADAR1 RNA Deaminase Limits Short Interfering RNA Efficacy in Mammalian Cells* 
The Journal of biological chemistry  2004;280(5):3946-3953.
Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.
doi:10.1074/jbc.M407876200
PMCID: PMC2947832  PMID: 15556947
16.  Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy 
Methods (San Diego, Calif.)  2006;39(4):316-328.
The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.
doi:10.1016/j.ymeth.2006.06.002
PMCID: PMC2442885  PMID: 16879979
Single molecule fluorescence; Nuclear transport; Particle tracking; Narrow-field epifluorescence microscopy; Cell imaging
17.  Chondrogenic Differentiation on Perlecan Domain I, Collagen II and Bone Morphogenetic Protein 2-Based Matrices 
Tissue engineering  2006;12(7):2009-2024.
Extracellular matrix (ECM) molecules in cartilage, cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). Our hypothesis was that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on two-dimensional P-C fibrils or three dimensional P-C/BMP-2-coasted (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II and aggrecan. Immunostaining for aggrecan, perlecan, tenascin and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We have concluded that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenenic differentiation.
doi:10.1089/ten.2006.12.2009
PMCID: PMC1774589  PMID: 16889529
Chondrogenesis; Perlecan; Bone Morphogenetic Protein-2 (BMP-2); Collagen II; Mesenchymal Cells; Tissue Engineering
18.  Nuclear import time and transport efficiency depend on importin β concentration 
The Journal of Cell Biology  2006;174(7):951-961.
Although many components and reaction steps necessary for bidirectional transport across the nuclear envelope (NE) have been characterized, the mechanism and control of cargo migration through nuclear pore complexes (NPCs) remain poorly understood. Single-molecule fluorescence microscopy was used to track the movement of cargos before, during, and after their interactions with NPCs. At low importin β concentrations, about half of the signal-dependent cargos that interacted with an NPC were translocated across the NE, indicating a nuclear import efficiency of ∼50%. At high importin β concentrations, the import efficiency increased to ∼80% and the transit speed increased approximately sevenfold. The transit speed and import efficiency of a signal-independent cargo was also increased by high importin β concentrations. These results demonstrate that maximum nucleocytoplasmic transport velocities can be modulated by at least ∼10-fold by the importin β concentration and therefore suggest a potential mechanism for regulating the speed of cargo traffic across the NE.
doi:10.1083/jcb.200605053
PMCID: PMC2064387  PMID: 16982803

Results 1-18 (18)