Plant Molecular Biology 50: 871–883, 2002.
© 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Imaging protein-protein interactions in living cells
Mark A. Hink
, Ton Bisseling
and Antonie J.W.G. Visser
MicroSpectroscopy Centre, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
author for correspondence; e-mail Ton.email@example.com);
Department of Structural Biology, Faculty
of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
Received 5 November 2001; accepted in revised form 23 June 2002
Key words: cells, FCS, ﬂuorescence spectroscopy, FRET, protein-protein interactions
The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and
the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment.
Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution
of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules.
Methods to visualize interaction can be based on FRET (ﬂuorescence detected resonance energy transfer), for
example in ﬂuorescence lifetime imaging microscopy (FLIM). Another method is based on ﬂuorescence correlation
spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether
a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein
interactions in vivo are reviewed.
Several elegant biochemical methods have been devel-
oped to study protein-protein interactions. In general,
such biochemical studies are carried out in vitro and
have provided valuable information about the proper-
ties of the studied molecules. However, to what extent
these properties reﬂect their behaviour in living cells
is not clear. The complex organization and the com-
partmentalization of plant cells make it probable that
molecular behaviour in the test tube and in the cell
are not identical and therefore it is essential to study
molecules in their natural environment.
Optical microscopy has been very useful to obtain
information about the sub-cellular location of proteins.
However, classical light microscopy, for example, can-
not reveal whether proteins interact with one another.
At best, optical microscopy can demonstrate that two
proteins occur in the same region within a cell. It
must be realised, however, that the spatial resolution
of light microscopy (about 300 nm in a standard con-
focal microscope) is orders of magnitude larger than
the average size of a protein molecule (diameter about
3 nm for a globular protein of 30 kDa). Therefore,
it is unclear whether molecules observed in the same
region in a light microscopic image interact or not.
For example, proteins located in the nucleus may co-
localize, but, of course, not all nuclear proteins interact
with each other. So how can interactions be imaged in
a living cell?
The integration of ﬂuorescence spectroscopy in
light microscopy adds a new dimension to microscopy
since in addition to spatial resolution now also infor-
mation about the molecular behaviour of molecules
can be obtained. Methods to visualize interaction can
be based on FRET (ﬂuorescence detected resonance
energy transfer), for example in ﬂuorescence lifetime
imaging microscopy (FLIM) by which the ﬂuores-
cence lifetime of a ﬂuorescent dye as a function of
intracellular space can be determined. Another method
is ﬂuorescence correlation spectroscopy (FCS) that
allows determining the diffusion rate of single mole-
cules, providing insight into whether a protein is part
of a larger complex or not. Here, both FRET- and FCS-
based approaches to study protein-protein interactions
in vivo are described.