in vivo

a
b
Recent publications
Figure 1
Molecular imaging enables one to visualize and quantify molecular dynamics, interactions, and kinetics in cells for molecular
systems biology. (a) HILO microscopy for molecular imaging in cells. Illumination by a highly-inclined and thin beam increases
image intensity and decreases background intensity, yielding a signal/background ratio up to about eightfold greater than
that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images. (b) To evaluate the
HILO microscopy technique, we reconstructed a three-dimensional image from serial images of nuclear pore complexes
labeled with EGFP-importin β in an MDBK cell. Scale bar, 5.0 µm.
molecules. During the first phase of development
we achieved single-color HILO, but now we have
installed a new multi-color system to observe
intermolecular interactions in ever greater detail.
Quantification of Molecular Kinetics and
Interactions in Cells
To explore potential new uses of this
technology, we performed quantitative analysis
on nuclear import to demonstrate its application
to kinetic studies (Fig. 2 left). We could visualize
single molecules of GFP-importin β mediating
the import of cargo through nuclear pores in
cells as bright spots on the nuclear envelope at a
concentration at or below the nanomolar range.
The interaction time of single molecules with
nuclear pore complexes (NPCs) was determined
base on the duration of the fluorescent spots.
At greater than nanomolar concentrations
of GFP-importin β, individual NPCs were clearly
visualized as fluorescent spots (Fig.1b). The
number of GFP-importin β molecules bound to
a single NPC could be estimated by determining
the ratio of the fluorescence intensities of single
NPC images to that of single molecule images.
We determined the number of bound molecules
as a function of the GFP-importin β concentration.
Importin β in the absence of RanGTP exhibited
two types of binding with the NPC. The higher
affinity binding showed a dissociation constant
of approximately 0.3 nM.The maximum number
of bound molecules was calculated to be
approximately 7 molecules/NPC, although the
actual number is probably 8 molecules/NPC
since the NPC exhibits an 8-fold symmetry. The
lower affinity binding exhibited almost the same
kinetic parameters irrespective of the presence or
absence of cargo, with a dissociation constant of
70+50/-30 nM and a maximum bound number
of 110 +60/-40 molecules / NPC.
We then examined the rate of translocation
into the nucleus of cargo or importin β at the
single-cell level. The rate of nuclear import was
obtained as the maximum slope in the time
course of nuclear accumulation. The import rate
was plotted as a function of the cargo-importin β
concentrations, and it was fitted to a Michaelis-
Menten equation with a Michaelis constant of 54
±16 nM. The import rate showed a significant
correlation with the inverse of the retention time,
which represents the frequency of translocations
per second of single molecules. The correlation
coefficient was approximately 8 molecules/NPC,
which exhibits the number of the translocation
sites into the nucleus per NPC. This corresponds
well with the 8-fold symmetry of the NPC
structure.
“In silico” Modeling and Simulation
As shown above, molecular interactions
with the assembled NPC were quantified by
single molecule analysis - retention times,
the number of associated molecules, the
dissociation constant, and stoichiometry of
import were all determined (Fig. 2 left). In order
to understand the molecular mechanism of
nuclear import, a numerical model of import was
constructed using these kinetic parameters.
Computer simulation was carried out based on
the model with two types of binding sites. The
simulation fit very well with both the results of
single-molecule experiments and the molecular
kinetic features in cells.
As demonstrated herein, the combination
of single molecule quantification and modeling
opens new approaches for developing molecular
system biology.
Tokunaga, M., Imamoto, N.,
Sakata-Sogawa, K.: Highly inclined
thin illumination enables clear
single-molecule imaging in cells.
Nat Methods. 5, 159-161 (2008).
Yamasaki, S., Sakata-Sogawa, K.,
Hasegawa, A., Suzuki, T., Kabu, K.,
Sato, E., Kurosaki, T., Yamashita,
S., Tokunaga, M., Nishida, K.,
Hirano, T.: Zinc is a novel second
messenger. J. Cell Biol. 177,
637-645 (2007)
Yamasaki S., Ishikawa, E.,
Sakuma, M., Ogata, K., Sakata-
Sogawa K., Hiroshima, M., Wiest,
D. L., Tokunaga M., Saito, T.
Mechanistic basis of pre-T cell
receptor-mediated autonomous
signaling critical for thymocyte
development. Nature Immunol. 7,
67-75 (2006)
Yokosuka T., Sakata-Sogawa K.,
Kobayashi, W., Hiroshima, M.,
Hashimoto-Tane, A., Tokunaga
M., Dustin, M. L., Saito, T.
Newly generated T cell receptor
microclusters initiate and sustain
T cell activation by recruitment
of Zap70 and SLP-76. Nature
Immunol. 6, 1253-1262 (2005)
Shiina N., Shinkura K., Tokunaga
M. A novel RNA-binding protein in
neuronal RNA granules: Regulatory
machinery for local translation.
J.Neuroscience, 25, 4420-4434
(2005)
a
b
Imaging
and
In Silico
Figure 2
The combination of single molecule
quantification and “in silico” modeling
opens new approaches for developing
molecular systems biology. (left) Molecular
interactions with the assembled NPC were
quantified by single molecule analysis.
Retention times, the number of associated
molecules, the dissociation constant,
and stoichiometry of import were all
determined. Simulation based on a model
with two types of multi-binding sites using
these parameters fit well with the molecular
kinetic features in cells. (right) Aiming at
understanding immune cells as molecular
systems, we are going to construct “in
silico” cell models based on single-molecule
quantification. Bidirectional research is
essential to reconstruct cell functions in
silico; research from molecules to systems
by single molecule analysis, and feedback
research from systems to molecules.
dynamics, interactions, and kinetics in cells
for molecular systems biology.
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