Analysis of the molecular dynamics of medaka nuage
proteins by fluorescence correlation spectroscopy and
fluorescence recovery after photobleaching
Issei Nagao
1,
*, Yumiko Aoki
2
, Minoru Tanaka
2
and Masataka Kinjo
1
1 Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan
2 Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology, Okazaki, Japan
In most animals, primordial germ cells (PGCs) develop
distinctly from other cell lineages at a very early
embryonic stage, migrate towards the prospective
gonadal area, and then differentiate into gametes in
the gonads. Formation of the PGC requires germ
plasm, which contains electron-dense structures called
nuages that are believed to contain the determinants of
germ cells [1,2]. Although the nuage was reported half
Keywords
fluorescence correlation spectroscopy;
fluorescence recovery after photobleaching;
medaka; primordial germ cell; vasa
Correspondence
M. Kinjo, Laboratory of Molecular Cell
Dynamics, Faculty of Advanced Life
Science, Hokkaido University, Kita 21
Nishi 11, Kita-ku, Sapporo 001-0021, Japan
Fax: +81 1 706 9006

cytosolic Olvas–GFP was also observed to have a diffusion movement of
7.0 lm
2
Æs
)1
. Interestingly, Olvas–GFP could be expressed in HeLa cells,
and formed granules that were similar to nuages in medaka PGCs. Olvas–
GFP also exhibited a constraint movement in the granules and diffused in
the cytosol of HeLa cells, just as in the medaka embryo. The other two
gene products, Nanos and Tudor of the medaka, which are known as con-
stituents of the nuage, could also be expressed in HeLa cells and formed
granules that colocalized with Olvas–GFP. Nanos–GFP and Tudor–GFP
exhibited constraint movement in the granules and diffused in the cytosol
of HeLa cells. These results suggest that these granules in the HeLa cell are
not simple aggregations or rigid complexes, but dynamic structures consist-
ing of several proteins that shuttle back and forth between the cytosol and
the granules.
Abbreviations
CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; FAF, fluorescence autocorrelation function; FCS, fluorescence correlation
spectroscopy; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; LSM, laser scanning microscopy;
PGC, primordial germ cell; RFP, red fluorescent protein.
FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS 341
a century ago, its roles and functions in animal germ
lines are poorly understood. Recently, it was reported
that, in Drosophila, the function of the nuage might be
related to the protection of the genome via repression
of the selfish genetic elements in the female germ line
[3]. The nuage is known to be an electron-dense struc-
ture; however, little is known about its dynamic prop-
erties of morphological change or component exchange

fluctuations of probes provides the diffusional proper-
ties of proteins [13] and binding interactions [14].
FRAP is a conventional technique used to study the
kinetic properties of proteins in a cell by measuring
the fluorescence recovery rate in a bleached area [20].
Unbleached molecules enter into the bleached area
from the outside, and the fluorescence intensity is
recorded by time-lapse microscopy. The recovery curve
provides qualitative and quantitative information such
as the diffusion constant and the amount of the mobile
fraction. Although FCS and FRAP also provide diffu-
sion properties of fluorescent molecules, these methods
can be taken to be complementary, because FCS is
well suited to fast processes occurring in microseconds
to milliseconds in the observation area, whereas FRAP
is preferable for slower processes that take from milli-
seconds to seconds [18,21,22].
Herein we report dynamic properties of proteins in
the PGC determined by FCS and FRAP. A fusion
protein consisting of Olvas and green fluorescent pro-
tein (GFP) (Olvas–GFP) expressed in the PGC forms
granules that exhibit an amorphous shape and time-
dependent morphological changes. The movements of
Olvas–GFP in the nuage and the cytosol were quite
different, suggesting that this protein interacted with a
cellular matrix such as the cytoskeleton or assembled
itself to form larger complexes. When the protein was
expressed in HeLa cells, Olvas–GFP formed distinct
granules that colocalized with Nanos or Tudor. In the
granules, these three proteins exhibit very characteristic

Olvas–GFP shuttles between the nuage
and cytosol
Next, we analyzed the diffusion of Olvas–GFP in the
PGCs prepared from the embryo, using FCS and
FRAP (Fig. 3). Movement of Olvas–GFP was
Dynamic nature of medaka nuage proteins I. Nagao et al.
342 FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS
measured outside of the nuage in the cytoplasm of the
migrating PGC. FCS analysis revealed that Olvas–
GFP diffused with a diffusion constant of 7.0 lm
2
Æs
)1
(Fig. 3A). During the measurement of the PGC, the
olvas
3'UTR
GFP/RFP 3'UTR
nanos
3'UTR
GFP-3'UTR
GFP/RFP-
olvas
tudor
3'UTR
GFP/RFP
GFP/RFP
GFP/RFP
GFP/RFP-
nanos
GFP/RFP-

1.6
2A
B
1 10 100 1000 10000 100000
Time (s)
Time (µs)
Normalized G(τ)
Relative intensity
Fig. 3. FCS and FRAP analyses of Olvas–GFP in the PGC. FCS was
used to measure the movement of Olvas–GFP into the cytosol out of
the nuage region of the PGC. Representative correlation curves are
shown (A). The measurement point is indicated by the cross-hair (+)
in the LSM image of Olvas–GFP transiently expressed in PGCs
(inset). The correlation curve of Olvas–GFP (squares) shifted to a
slower part as compared to GFP (diamonds) only. In the cytosol,
Olvas–GFP diffused at D = 7.0 lm
2
Æs
)1
. FRAP analysis was per-
formed in the nuage region (B). The curve is the mean of three inde-
pendent measurements. The bleached position is indicated by the
white circle (inset). FRAP curve analysis shows that Olvas–GFP
moves slowly at D = 0.15 lm
2
Æs
)1
and D = 0.01 lm
2
Æs

To investigate the mobility of Olvas–GFP in detail,
we performed in vitro analysis using HeLa cells. A
fusion gene was constructed with the cytomegalovirus
(CMV) promoter and simian virus 40 poly(A) signal.
Surprisingly, Olvas–GFP formed granules in the
cytoplasm (Fig. 4). To verify that these granules were
not merely the simple aggregates often seen in trans-
fected cultured cells, a nanos–RFP or tudor–GFP
fusion gene was cotransfected with the olvas–GFP or
olvas–RFP fusion gene, and diffusion analysis by
FCS and FRAP was performed. As shown in
Fig. 4A, Olvas–GFP and Nanos–RFP colocalized on
the granules in the cytoplasm, and similarly, Olvas–
RFP shared the granules with Tudor–GFP (Fig. 4B).
Next, we carried out FCS and FRAP analyses to
determine the mobility of Olvas–GFP, Nanos–GFP
and Tudor–GFP in HeLa cells (Fig. 5). FCS
measurement revealed that these three proteins dif-
fused with diffusion constants of 11.7, 12.9 and
5.4 lm
2
Æs
)1
, respectively, in the part of the cytoplasm
outside of the granules. FRAP analysis in the gran-
ules provided typical recovery curves of these fusion
proteins: a diffusion constant with two components
of 0.9 and 0.03 lm
2
Æs

B
Fig. 4. Olvas, Nanos and Tudor fusion proteins expressed in the
HeLa cell form granules. olvas–GFP and nanos–RFP (A), and olvas–
RFP and tudor–GFP (B), were cotransfected into HeLa cells. LSM
images of the HeLa cells are presented. These proteins formed
granules in the cytoplasm. Olvas–GFP and Nanos–RFP, and Olvas–
RFP and Tudor–GFP, are colocalized in the granules.
Dynamic nature of medaka nuage proteins I. Nagao et al.
344 FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS
into HeLa cells. Eight conserved motifs of the olvas
gene [18] are depicted in black boxes in Fig. 6B. The
del1, del2 and del3 mutants lack the two N-terminal
motifs, six N-terminal motifs, and two C-terminal
motifs, respectively. All three deletion series of proteins
were uniformly present in the cytoplasm in large popu-
lations of transfected cells (Fig. 6B, upper panels).
However, in a small number of transfected cells, fluo-
rescent granules were found in the cytoplasm (Fig. 6B,
lower panels). FCS analysis revealed that all deletion
mutants had diffusion constants ranging from 10.5 to
11.3 lm
2
Æs
)1
in the cytosol (Fig. 7A). In contrast,
FRAP analysis revealed that Olvas–GFP deletion pro-
teins were almost all immobilized in the granules
(Fig. 7B), clearly indicating that these granules could
be discriminated from the granules observed in
Fig. 4B. These granules containing Olvas deletion

Olvas–GFP shows characteristic movement in both
the nuages of PGCs of medaka embryos and the gran-
ules in HeLa cells. FRAP revealed that it moved with
two diffusion components in both PGCs and HeLa
cells: 0.15 and 0.01 lm
2
Æs
)1
in PGCs, and 0.9 and
0.03 lm
2
Æs
)1
in HeLa cells. The observation of two
components here indicates that more than two compo-
nents or architectures are involved in the formation
of the granules. Such multicomponents have been
observed in the P-body and stress granule [26]. In the
cytosol of both PGCs and HeLa cells, diffusing protein
was observed. The other two components of the nuage,
Nanos and Tudor, exhibit diffusion constants of 1.7
0
0.2
0.4
0.6
0.8
1
1.2
0 10203040
0

and D = 5.4 lm
2
Æs
)1
, respectively. FRAP analy-
ses of Olvas–GFP, Nanos–GFP and Tudor–GFP were performed for
the granule (B). Each curve is the mean of 10 independent mea-
surements. The bleached position is indicated by the white circle
(inset). These recovery curves show diffusion constants
D = 0.9 lm
2
Æs
)1
and D = 0.03 lm
2
Æs
)1
for Olvas–GFP (diamonds),
D = 1.7 lm
2
Æs
)1
for Nanos–GFP (squares), and D = 0.16 lm
2
Æs
)1
for Tudor–GFP (triangles).
I. Nagao et al. Dynamic nature of medaka nuage proteins
FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS 345
and 0.16 lm

dependent on the complete set of the DEAD-box
motifs in olvas. These results indicate that the granules
are not merely artificial aggregates, thought to be the
result of protein misfolding, but might reflect the nat-
ure of the nuage in the PGC.
Recently, there have been some reports that germline-
specified microRNAs are essential in germ cell develop-
ment [27]. Vasa is also thought to interact with Piwi and
Aubergine, which are members of the AGO protein
family [28,29], suggesting that the nuage is implicated in
the Piwi-interacting RNA pathway. It has been shown
that the nuage contains RNAs and proteins that may
have important roles in the development of PGCs [1–3].
It is interesting that rapid exchange of nuage compo-
nents occurred, because such exchange suggests that the
nuage is not only a static storage site, but also a
dynamic RNA- and protein-processing particle. In this
sense, our finding that cultured HeLa cells expressed
Olvas, Nanos and Tudor provides a very attractive
system with which to investigate the features of PGCs.
Although these tests were carried out in HeLa cells
only, they could potentially be applied to other types of
cultured cells.
del1 del2 del3
GFP
Olvas-GFP
GFP
GFP
del1
del2

The coding sequences of olvas, nanos and tudor were
modified by PCR with BglII and EcoRI, using primers
5¢-GGAGATCTAAAATGGACGACTGGGAGGAAGA-3¢
and 5¢-GCGAATTCGTTGAAAACTTTTAATTATCA
GGAGAAAAC-3¢,5¢-CGAGATCTAGCATGTCAGACG
TGGAGTCTGGA-3¢ and 5¢-GCGAATTCGCAACCAAA
GACAACCTGGTTTTAATGTTTTGA-3¢, and 5¢-CGAG
ATCTGAAATGAACGAGCTGCGTATGCCGAA-3¢ and
5¢-GCG AATTCAAC ACAAGAG TTGT TTTATAT TGAA
CCCA-3¢, respectively. The PCR product was digested
and ligated into the multiple cloning site of pEGFP-Cl
(Clontech, Palo Alto, CA, USA) or mRFP [30]. This plas-
mid encoded fluorescent protein and Olvas, Nanos or
Tudor fusion proteins [enhanced GFP (EGFP)–Olvas,
mRFP–Olvas, EGFP–Nanos, mRFP–Nanos, EGFP–
Tudor, and mRFP–Tudor chimera], and was transcribed
from the CMV promoter.
In vitro RNA synthesis and microinjection
The olvas–GFP described above was employed as a
template for PCR, using primers 5¢-GCGCTAGCTAAT
ACGACTCACT ATAG GGA GATC TAAA ATGG AC GAC
TGGGAGGAAGA-3¢ and 5¢-GCGAATTCGTTGAAA
ACTTTTAATTATCAGGAGAAAAC-3¢. This PCR frag-
ment has a T7 promoter for RNA synthesis. Capped RNA
was synthesized by T7 RNA polymerase, using an mMes-
sage mMachine T7 Kit (Ambion, Inc., Austin, TX, USA).
No poly(A) tail was added. Finally, 100 ngÆlL
)1
RNA was
injected into a one-cell embryo.

0.5
1
1.5
2
1 10 100 1000 10 000 100 000
0
0.2
0.4
0.6
0.8
1
1.2
B
A
0 10203040
Normalized G(τ)Relative intensity
Time (μs)
Time (s)
Fig. 7. FCS and FRAP analyses of Olvas deletion series in HeLa
cells. Deletion series diffusion was measured by FCS in the cytosol
outside the region of the granule. Representative correlation curves
are shown (A). FCS analysis of Olvas deletion series revealed that
all these proteins diffused in the cytosol at D = 11.3 lm
2
Æs
)1
(del1;
diamonds), D = 10.5 lm
2
Æs

oto’s Ringer solution containing 3.5 mm 1-heptanol (3.5 m
stock solution; Wako, Osaka, Japan). Cultured cells were
washed with phenol red-free Opti-MEM I reduced-serum
medium (Invitrogen, Carlsbad, NM, USA) twice to remove
phenol red dye; then the medium was replaced by Opti-
MEM I. Immediately thereafter, FCS measurements were
carried out. The obtained FAF was fitted by a one-compo-
nent, two-component or three-component model (i = 2 or 3
in the following equation) as follows:
G sðÞ¼
ItðÞItþ sðÞ
hi
I
hi
2
¼ 1 þ
1
N
X
i
Fi 1 þ
s
si

À1
1 þ
s
s
2
si

)6
cm
2
Æs
)1
), and diffusion
times s
Rh6G
and s
sample
were obtained as the following
equation:
D
sample
D
Rh6G
¼
s
Rh6G
s
sample
FRAP analysis
FRAP measurements were performed on the same setup of
the laser scanning microscope as used for FCS analysis.
The detection gain was adjusted to the fluorescence of the
GFP fusion proteins almost at the saturation level of the
detector, and the pinhole was opened widely enough to
acquire fluorescence from the cell. Ten single scans were
acquired, followed by four bleach pulses without scanning.
Single section images were collected at 0.2 s intervals.

University, Japan) for technical advice on the FRAP
experiment. This research was supported by the 21st
Century COE Program for ‘Advanced Life Science on
the Base of Bioscience and Nanotechnology’ in Hok-
kaido University. This research was partly supported
by Grands-in-Aid for Scientific Research (A) 18207010
from JSPS, and Grants-In-Aid for Scientific Research
(Kakenhi) ‘Nuclear Dynamics (17050001)’ by the
Ministry of Education, Culture, Sports, Science and
Technology of Japan (to M. Kinjo).
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