Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging

PLoS One. 2018 Apr 25;13(4):e0195825. doi: 10.1371/journal.pone.0195825. eCollection 2018.

Abstract

Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism*
  • Epitopes / metabolism
  • HEK293 Cells
  • Hemagglutinins / chemistry
  • Humans
  • Image Processing, Computer-Assisted
  • Microscopy
  • Molecular Imaging / methods*
  • Protein Transport
  • Recombinant Fusion Proteins / metabolism
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Time Factors

Substances

  • Epitopes
  • Hemagglutinins
  • Recombinant Fusion Proteins
  • ATP1A1 protein, human
  • Sodium-Potassium-Exchanging ATPase

Associated data

  • Dryad/10.5061/dryad.kc42g

Grants and funding

This work was supported by Swedish Research Council www.vr.se 2013-6041 to H.B., Swedish Research Council www.vr.se 2015-4198 to HJ.B., Swedish Research Council www.vr.se 2016-00968 to HJ.B., Swedish foundation for strategic research www.stratresearcg.se RIF14-0091 to H.B Märta och Gunnar V. Philipsons foundation. to HJ.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.