Monitoring the diffusion behavior of Na,K-ATPase
by fluorescence correlation spectroscopy (FCS)
upon fluorescence labelling with eGFP or
Dreiklang

• Authors: Cornelia Junghans , Franz-Josef Schmitt , Vladana Vukojević , Thomas Friedrich
• Languages of publication: EN

Abstracts

EN

Measurement of lateral mobility of membraneembedded
proteins in living cells with high spatial and
temporal precision is a challenging task of optofluidics.
Biological membranes are complex structures, whose
physico-chemical properties depend on the local lipid
composition, cholesterol content and the presence of integral
or peripheral membrane proteins, which may be involved
in supramolecular complexes or are linked to cellular
matrix proteins or the cytoskeleton. The high proteinto-
lipid ratios in biomembranes indicate that membrane
proteins are particularly subject to molecular crowding,
making it difficult to follow the track of individual
molecules carrying a fluorescence label. Novel switchable
fluorescence proteins such as Dreiklang [1], are, in principle,
promising tools to study the diffusion behavior of individual
molecules in situations of molecular crowding due
to excellent spectral control of the ON- and OFF-switching
process. In this work, we expressed an integral membrane
transport protein, the Na,K-ATPase comprising the human
α2-subunit carrying an N-terminal eGFP or Dreiklang tag
and human β1-subunit, in HEK293T cells and measured
autocorrelation curves by fluorescence correlation spectroscopy
(FCS). Furthermore,we measured diffusion times
and diffusion constants of eGFP and Dreiklang by FCS,
first, in aqueous solution after purification of the proteins
upon expression in E. coli, and, second, upon expression
as soluble proteins in the cytoplasm of HEK293T cells. Our
data show that the diffusion behavior of the purified eGFP
and Dreiklang in solution as well as the properties of the
proteins expressed in the cytoplasm are very similar. However,
the autocorrelation curves of eGFP- and Dreiklanglabeled
Na,K-ATPase measured in the plasma membrane
exhibit marked differences, with the Dreiklang-labeled
construct showing shorter diffusion times. This may be related
to an additional, as yet unrecognized quenching process
that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1–
100 ms). Since the origin of this quenching process is currently
unclear, care has to be taken when the Dreiklang label
is intended to be used in FCS applications.

Keywords

EN

Membrane protein diffusion; solvent viscosity; diffusion constants; lateral mobility; molecular crowding;HEK 293 cells; transient transfection; photoswitchable; fluorescence proteins

• Publisher: De Gruyter Open
• Journal: Optofluidics, Microfluidics and Nanofluidics
• Year: 2015
• Volume: 2
• Issue: 1

Dates

accepted

20 - 10 - 2015

online

31 - 12 - 2015

received

6 - 10 - 2015

Contributors

author

Cornelia Junghans

• Technical University of
  Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
  Berlin, Germany

author

Franz-Josef Schmitt

• Technical University of
  Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
  Berlin, Germany

author

Vladana Vukojević

• Karolinska Institutet, Department of Clinical
  Neuroscience, Center for Molecular Medicine CMM L8:01, 17176
  Stockholm, Sweden

author

Thomas Friedrich

• Technical University of
  Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
  Berlin, Germany

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Identifiers

DOI

10.1515/optof-2015-0001