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Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules - PubMed

  • ️Thu Jan 01 2015

Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules

Moumita Ray et al. Bioconjug Chem. 2015.

Abstract

We describe a method for quantitative monitoring of subcellular protein trafficking using nanoparticle-stabilized nanocapsules for protein delivery. This method provides rapid delivery of the protein into the cytosol, eliminating complications from protein homeostasis processes found with cellularly expressed proteins. After delivery, nuclear protein trafficking was followed by real time microscopic imaging. Quantitative analyses of the accumulation percentage and the import dynamics of the nuclear protein trafficking, demonstrate the utility of this method for studying intracellular trafficking systems.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1

Delivery of eGFP fused with nuclear localization signals (NLS) to cells using NPSCs. (a) Schematic representing the cytosolic delivery and nuclear accumulation of proteins with NLSs. (b) Structure of eGFP fused with NLSs. (c) LSCM images showing different cellular distribution patterns of eGFP fused with NLSs. Bars: 20 μm. (d) Analysis revealing the increase in nuclear intensities of NLS-eGFPs compared to that in the cytosol of the same cell (6 cells per group).

Figure 2
Figure 2

Nuclear import of eGFP fused with NLS is rapid and ATP dependent. (a) Time-lapse fluorescent microscopic images show nuclear accumulation of NLSc-Myc-eGFP starts within 1 minute after presenting in cytosol. (b,c) Time-lapse LSCM images unveil the kinetics of nuclear import of NLSc-Myc-eGFP. (d) No nuclear accumulation of NLSc-Myc-eGFP is observed after the delivery following ATP depletion in the presence of 3 mg/mL NaN3 and 50 mM 2-deoxyglucose. Bars: 20 μm.

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