Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Quantum dot assisted tracking of the intracellular protein Cyclin E in Xenopus laevis embryos.
Brandt YI, Mitchell T, Smolyakov GA, Osiński M, Hartley RS.
???displayArticle.abstract???
Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells. We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time. These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.
Figure 1. Transmission electron microphotographs of the synthesized CdSe-ZnS QD. (a) Scale bar is 50 nm. (b) Scale bar is 20 nm. (c) Scale bar is 10 nm.
Figure 2. Schematic showing experimental design. (a) DHLA capping of CdSe-ZnS (QD564) and subsequent conjugation of (His6)-Cyclin E (modified from [5]). (b) Microinjection of (QD564)-His6Cyclin E into 2-cell Xenopus embryos and (c) confocal imaging of microinjected pre-MBT and MBT Xenopus embryos. In (a) the DHLA molecule contains a bidentate thiol moiety on one end, allowing its stable attachment to the inorganic QD surface. Coupling of (His6)-Cyclin E to QD is achieved via strong metal affinity between the histidine tag of the protein and Zn+2 atoms on the QD surface. The schematic is not to scale.
Figure 3. Localization of (QD564)-His6Cyclin E in live pre-MBT (4 hpf, 64-cell embryo, a-c) and MBT (6 hpf, 2048-cell embryo, d-f) Xenopus laevis embryos. One cell of embryos at the 2-cell stage was microinjected with (QD564)-His6Cyclin E and visualized using confocal microscopy. (a, d) fluorescence channel; (b, e) light channel; (c, f) merged fluorescence and light channels. Nuclei are marked with white arrowheads in panels b, f. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected and viewed in at least 3 separate experiments.
Figure 4. Localization of exogenous Cyclin E in pre-MBT and MBT Xenopus laevis embryos. One cell of 2-cell embryo was microinjected with in vitro transcribed Myc6−GFP-Cyclin E RNA, collected at indicated time points, and the translated protein detected in fixed and stained embryos. For immunofluorescence analysis of Cyclin E localization, embryos were collected at 4 hpf, pre-MBT (a-c) or at 6 hpf, MBT (d-f). (a, d) Embryos were fixed and stained with an antibody against the Myc6 tag (αMyc) followed by an Alexa488 conjugated secondary antibody. (b, e) Embryos were counterstained with DAPI to visualize the nuclei. (c, f). Merged image of the Alexa488 and DAPI. White arrowheads in d-f indicate nuclei. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. Scale bars are 100 μM. At least 20 embryos were injected in at least 3 separate experiments, with at least 5 embryos fixed per timepoint for analysis.
Figure 5. Cyclin E accumulates in the nucleus of live Xenopus laevis embryos at the MBT (6 hpf). One cell of the 2-cell embryo was microinjected with in vitro transcribed Myc6-GFP-Cyclin E RNA and the translated protein visualized in live embryos using confocal microscopy in real time. (a, b) Fluorescence channel, Z stack images #5 and #8 from the top, respectively. Nuclei are marked with white arrowheads. (c) Light field image. A 3D image is shown. Scale bars are 100 μM. Embryos were viewed with a 10X objective on a Zeiss LSM 510 confocal microscope equipped with a META detector, and analyzed using LSM510 Image Acquisition software. At least 20 embryos were injected in at least 3 separate experiments.
Andrews,
Actin restricts FcepsilonRI diffusion and facilitates antigen-induced receptor immobilization.
2008,
Pubmed Bakalova,
Silica-shelled single quantum dot micelles as imaging probes with dual or multimodality.
2006,
Pubmed Boeneman,
Sensing caspase 3 activity with quantum dot-fluorescent protein assemblies.
2009,
Pubmed Bouzigues,
Single quantum dot tracking of membrane receptors.
2007,
Pubmed Brandt,
Developmental downregulation of Xenopus cyclin E is phosphorylation and nuclear import dependent and is mediated by ubiquitination.
2011,
Pubmed
,
Xenbase Chan,
Quantum dot bioconjugates for ultrasensitive nonisotopic detection.
1998,
Pubmed Chevalier,
Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication.
1996,
Pubmed
,
Xenbase Clapp,
Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins.
2006,
Pubmed Dahan,
Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking.
2003,
Pubmed D'Alessio,
Nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments in Schizosaccharomyces pombe.
2003,
Pubmed Delehanty,
Delivering quantum dots into cells: strategies, progress and remaining issues.
2009,
Pubmed Dubertret,
In vivo imaging of quantum dots encapsulated in phospholipid micelles.
2002,
Pubmed
,
Xenbase Dutta,
Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.
1995,
Pubmed Feilmeier,
Green fluorescent protein functions as a reporter for protein localization in Escherichia coli.
2000,
Pubmed Foresti,
Protein domains involved in assembly in the endoplasmic reticulum promote vacuolar delivery when fused to secretory GFP, indicating a protein quality control pathway for degradation in the plant vacuole.
2008,
Pubmed Gao,
In vivo cancer targeting and imaging with semiconductor quantum dots.
2004,
Pubmed Goldman,
Fluoroimmunoassays using antibody-conjugated quantum dots.
2005,
Pubmed Guthrie,
Patterns of junctional communication during development of the early amphibian embryo.
1988,
Pubmed
,
Xenbase Hainfeld,
Ni-NTA-gold clusters target His-tagged proteins.
1999,
Pubmed Hwang,
Cyclin E in normal and neoplastic cell cycles.
2005,
Pubmed Jackman,
Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm.
2002,
Pubmed Jaiswal,
Use of quantum dots for live cell imaging.
2004,
Pubmed Ji,
High-efficient energy funneling based on electrochemiluminescence resonance energy transfer in graded-gap quantum dots bilayers for immunoassay.
2014,
Pubmed Keyomarsi,
Cyclin E, a potential prognostic marker for breast cancer.
1994,
Pubmed Koole,
Paramagnetic lipid-coated silica nanoparticles with a fluorescent quantum dot core: a new contrast agent platform for multimodality imaging.
2008,
Pubmed Landgraf,
Segregation of molecules at cell division reveals native protein localization.
2012,
Pubmed Lee,
Quantitative molecular profiling of biomarkers for pancreatic cancer with functionalized quantum dots.
2012,
Pubmed Lingerfelt,
Preparation of quantum dot-biotin conjugates and their use in immunochromatography assays.
2003,
Pubmed Liu,
Application of ZnO quantum dots dotted carbon nanotube for sensitive electrochemiluminescence immunoassay based on simply electrochemical reduced Pt/Au alloy and a disposable device.
2014,
Pubmed Lu,
Aqueous synthesized near-infrared-emitting quantum dots for RGD-based in vivo active tumour targeting.
2013,
Pubmed Medintz,
Quantum dot bioconjugates for imaging, labelling and sensing.
2005,
Pubmed Medintz,
Self-assembled nanoscale biosensors based on quantum dot FRET donors.
2003,
Pubmed Newport,
Regulation of the cell cycle during early Xenopus development.
1984,
Pubmed
,
Xenbase Peng,
Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor.
2001,
Pubmed Sive,
Xenopus laevis In Vitro Fertilization and Natural Mating Methods.
2007,
Pubmed
,
Xenbase Sive,
Microinjection of Xenopus embryos.
2010,
Pubmed
,
Xenbase Stylianou,
Imaging morphogenesis, in Xenopus with Quantum Dot nanocrystals.
2009,
Pubmed
,
Xenbase Takagi,
Transgenic Xenopus laevis for live imaging in cell and developmental biology.
2013,
Pubmed
,
Xenbase Trombetta,
Quality control and protein folding in the secretory pathway.
2003,
Pubmed Vitale,
Recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor.
2005,
Pubmed Wang,
Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.
2014,
Pubmed Weber,
The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities.
2004,
Pubmed
,
Xenbase Yi,
Silica-coated nanocomposites of magnetic nanoparticles and quantum dots.
2005,
Pubmed Zhang,
Single-cell microinjection technologies.
2012,
Pubmed Zhelev,
Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers.
2006,
Pubmed Zhu,
EGFP tags affect cellular localization of ATP7B mutants.
2013,
Pubmed
