Brandt YI et al. (2015), Quantum dot assisted tracking of the intracellu...

XB-ART-50554

J Nanobiotechnology 2015 Apr 29;13:31. doi: 10.1186/s12951-015-0092-6.

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Quantum dot assisted tracking of the intracellular protein Cyclin E in Xenopus laevis embryos.

Brandt YI , Mitchell T , Smolyakov GA , Osiński M , Hartley RS .

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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.

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	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.

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