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 Tel: +81 1 706 9005 E-mail: kinjo@imd.es.hokudai.ac.jp *Present address Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST) and Japan Biological Informatics Consortium (JBIC), Tokyo, Japan Database DNA data bank of Japan accession numbers: olvas, AB063484; nanos3, AB306931; tudor, AB306932 (Received 27 June 2007, revised 13 November 2007, accepted 21 November 2007) doi:10.1111/j.1742-4658.2007.06204.x The nuage is a unique organelle in animal germ cells that is known as an electron-dense amorphous structure in the perinuclear region. Although the nuage is essential for primordial germ cell (PGC) determination and devel- opment, its roles and functions are poorly understood. Herein, we report an analysis of the diffusion properties of the olvas gene product of the medaka fish (Oryzias lapites) in PGCs prepared from embryos, using fluo- rescence correlation spectroscopy and fluorescence recovery after photo- bleaching. Olvas–green fluorescent protein (GFP) localized in granules thought to be nuages, and exhibited a constraint movement with two-com- ponent diffusion constants of 0.15 and 0.01 lm 2 Æs )1 . On the other hand, 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 in the cytosol in the living cell. The nuage is composed of large riboprotein complexes, and several proteins, such as Vasa, Nanos and Tudor, have been identified as important components. In Drosophila, these compo- nents are essential for formation of the PGC [4], and are thought to be involved in some aspect of transla- tion in germ cells [5–7]. In the teleost fish medaka (Oryzias latipes), Nanos and Olvas (Vasa homologs), are expressed in PGCs in the early embryonic stages [8–11], and are localized in granule-like structures in the cytoplasm of the PGC (Y. Aoki, I. Nagao, D. Saito, Y. Ebe, M. Kinjo & M. Tanaka, unpub- lished results). As a result of the recent progress in fluorescence imaging methods and microscope technology, it has become easy to visualize the localization of fluorescent- ly tagged proteins, to quantitate their abundance, and to investigate their dynamic properties such as mobility and interactions. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are often used to assess the dynamics and kinetic properties of proteins in living cells [12–19]. FCS detects the fluctuations of fluorescent intensity derived from the movement of a single fluorescent molecule in a very tiny observation area, which is defined by the diffraction limit of a laser beam and the volume of which is about 0.25 · 10 )15 L. The fluores- cence autocorrelation function (FAF) calculated from 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 movements, suggesting that the formation of the gran- ules is not merely an artificial phenomenon, but that it could be used for investigation of the features of PGCs. Results Time-lapse laser scanning microscopy (LSM) image analysis of Olvas reveals the dynamic nature of the nuage Previous studies showed that the 3¢-UTR of genes for nuage components was essential for germ cell-specific expression of the components [10,11]. Figure 1 shows a schematic diagram of the Olvas–GFP fusion con- structs used in this study. RNA transcribed from these constructs in vitro was injected into medaka eggs in the one-cell stage to visualize the localization and to mea- sure the mobility of the components in the PGCs. The medaka embryo was peeled off the chorion, and the segment containing the part of PGCs was excised for observation by microscopy and FCS measurements (Fig. 2A). We performed time-lapse 3D LSM analysis of Olvas–GFP in migrating PGCs. Olvas–GFP was observed at 0, 1, 2, 3, 4 and 9 min in the migrating PGCs at stage 24 (Fig. 2B). It was localized as differently sized granules in the cytoplasm, and the granules occupied a large volume in the cell, as seen in the zebrafish [23]. Time-lapse LSM observation revealed the morphological changes of nuage structure, such as one granule combining with another and one dividing into two or more parts (arrowhead in Fig. 2B). 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- tudor Fig. 1. Schematic diagram of the constructs microinjected into the medaka eggs and transfected into HeLa cells. The olvas, nanos and tudor coding sequences were joined to the C-terminal region of the GFP or RFP coding sequence in an in-frame manner. These fusion genes were derived from the T7 promoter and CMV promoter in the case of in vitro transcription and in the case of HeLa cells, respectively. Glass-bottom plate Medaka embryo Lateral plate mesoderm 0 min 1 min 2 min 3 min 4 min 9 min B A Fig. 2. Time-lapse LSM analysis of Olvas–GFP reveals the dynamic nature of the nuage. Schematic diagram of the preparation of PGCs of medaka specimen (A). Olvas–GFP was expressed in the medaka PGC at stage 24 and localized in the nuage. 3D imaging of Olvas– GFP was performed at the indicated times (B). The nuage was seen around the nucleus and came together and apart in this time scale. Arrowheads show the assembly and dissociation points. 0 0.2 0.4 0.6 0.8 1 1.2 0 5 10 15 20 0 0.4 0.8 1.2 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 )1 . 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 343 specimen was alive and moved slowly; the FCS mea- surement was done in 3 s, which is a short time as compared to usual FCS measurement. Such a short period of FCS measurement caused the correlation curve to be noisy. This diffusion constant was smaller than that calculated, as the molecules moved com- pletely free from cellular interactions, such as mono- mer and ⁄ or oligomer tandem GFP, thought to be a noninteractive protein in the HeLa cell [24], suggesting the existence of some interactive cellular partner. FRAP analysis of the nuage revealed the slow recovery of Olvas–GFP (Fig. 3B). When the whole part of the single compartment of nuage was bleached, slow recovery of the fluorescence was observed, indicating that Olvas–GFP was provided from the cytosol. The obtained curve indicated that Olvas–GFP has two dif- fusion constant components, 0.15 and 0.01 lm 2 Æs )1 . These results indicate that Olvas–GFP shuttles between the nuage and the cytosol. GFP and red fluorescent protein (RFP) fusion proteins of Olvas, Nanos and Tudor form granules in transfected HeLa cells 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 )1 in Olvas–GFP, a diffusion constant of 1.7 lm 2 Æs )1 in Nanos–GFP, and a diffu- sion constant of 0.16 lm 2 Æs )1 in Tudor–GFP. These results suggest that these granules are not simply artificial aggregations, but might have some features of the nuage in the PGC. Deletion analysis of Olvas–GFP indicates that the DEAD-box motif might play a role in dynamic properties The vasa gene is known to encode a putative RNA helicase and to have a DEAD-box motif [25]. To examine the involvement of the DEAD-box motif in protein mobility, we constructed Olvas–GFP deletion mutants (Fig. 6A), and transfected these constructs Olvas-GFP / Nanos-RFP Merge A Tudor-GFP / Olvas-RFP Mer g e 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 mutants might have been artificial aggregations, which are sometimes seen with overexpression in cultured cells. Once freely moving Olvas deletion molecules formed such an aggregation, they would be fixed in it, and not have a functioning shuttle mechanism, like native Olvas. This result indicates that the domains including a complete set of DEAD-box motifs are important in localizing the granules and in dynamic protein mobility. Discussion Herein we report the dynamic nature of Olvas–GFP expressed in medaka embryos and HeLa cells. Time- lapse LSM image analysis of the Olvas–GFP distribu- tion reveals that the shape of the nuage changes in a matter of minutes in migrating PGCs. Moreover, diffu- sion analysis reveals that Olvas–GFP remains in the nuage for seconds, and that Olvas–GFP in the cytosol diffuses rather freely. Although the nuage has been analyzed as an important structure for the formation and maintenance of germ cells [1,2], this is the first report that rapid protein exchange occurs in the cyto- sol and nuage in the germ cell. This may imply that the constituents of the nuage are changed and replaced during the developmental stages. We observed that Olvas–GFP expressed in HeLa cells also formed granules that were similar to nuages in medaka PGCs. Furthermore, the colocalization of Nanos–RFP or Tudor–GFP with the Olvas fusion gene strongly suggests that molecular interaction with each protein occurred in the granules. 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 0.2 0.4 0.6 0.8 1 1.2 1.4 A B 1 10 100 1000 10000 100 000 Time (s) Normalized G(τ) Relative intensity Time (µs) Fig. 5. FCS and FRAP analyses of Olvas–GFP, Nanos–GFP and Tudor–GFP in HeLa cells. Diffusion of Olvas–GFP, Nanos–GFP and Tudor–GFP was measured by FCS in the cytosol outside the region of the granule. Representative correlation curves are shown (A). Measurement points are indicated by the cross-hair (+) in the LSM image of Olvas–GFP transiently expressed in a HeLa cell (inset). These curves for Olvas–GFP (diamonds), Nanos–GFP (squares) and Tudor–GFP (triangles) exhibit diffusion constants D = 11.7 lm 2 Æs )1 , D = 12.9 lm 2 Æs )1 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 2 Æs )1 , respectively, in granules of HeLa cells when observed using FRAP. In the cytosol of HeLa cells, diffusing Nanos and Tudor proteins were also observed by FCS. Their diffusion constants were 12.9 and 5.4 lm 2 Æs )1 , respectively. FCS and FRAP can be considered as complementary techniques, as FRAP can be employed to examine slow processes of replace- ment of molecules in granules from other parts of the same granules or from the cytosol. We performed a bleaching experiment in a whole part of a single com- partment of the granule, followed by fluorescence recovery. The fluorescence recovery suggests that the nonbleached GFP fusion proteins in the granule are replaced from the cytosol. These results show that Olvas–GFP, Nanos–GFP and Tudor–GFP shuttle between the granules and the cytosol, and that exchange within the granule might also occur; however, we cannot discriminate between the two possible modes of recovery, replacement from the cytosol, and replace- ment through the cytosol from other granules. Deletion analyses of Olvas–GFP show that all dele- tion mutants are defective in formation of the functional granules, indicating that their formation is 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 385 278 6171 del3 GFP 489 1 B A Fig. 6. Expression of Olvas deletion series in HeLa cells. Schematic diagrams of Olvas deletion series constructs are shown (A). The numbers of amino acid sequences are presented above each drawing. These cod- ing sequences were derived from the CMV promoter. Eight conserved regions are indi- cated in black boxes. (B) LSM image of Olvas deletion series in HeLa cells. The deletion series constructs were transfected into HeLa cells, and the LSM images observed are presented. In some cells, there are granules that are thought to be aggregations. Dynamic nature of medaka nuage proteins I. Nagao et al. 346 FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS Experimental procedures Plasmid construction cDNA cloning by RT-PCR amplification of olvas, nanos and tudor coding sequences from Oryzias latipes was described elsewhere [8,10] (Aoki et al., unpublished results). 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. Cell culture and transfection with plasmid DNA HeLa cells were grown in a 5% CO 2 humidified atmosphere at 37 °C in DMEM supplemented with 10% fetal bovine serum, 2 · 10 5 UÆL )1 penicillin G, and 200 mgÆL )1 strepto- mycin sulfate. Cells were propagated every 1 or 2 days. For transient expression, cells were plated at a confluence of 10–20% on LAB-TEK chambered coverslips with eight wells (Nalge Nunc International, Naperville, IL, USA) for 12 h before transfection. DMEM (20 lL) and FuGENE 6 (1.2 lL; Roche Molecular Biochemicals, Mannheim, Ger- many) were mixed. Five minutes after mixing, 0.4 lg of the Olvas–GFP, Nanos–GFP and Tudor–GFP or Olvas–RFP, Nanos–RFP and Tudor–RFP fusion protein-encoding plas- mid DNAs was added to the prediluted FuGENE 6 solu- tion. The DNA solution was left for 15 min, and added to one well 12 h before FCS measurement. Microscopy Live-cell fluorescence microscopy was performed using an LSM510 inverted confocal laser scanning microscope (LSM; Carl Zeiss, Jena, Germany). EGFP was excited at the 488 nm laser line of a CW Ar+ laser, and mRFP was excited at the 0 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 )1 (del2; squares), and D = 10.9 lm 2 Æs )1 (del3; triangles), respectively. FRAP analyses of Olvas deletion ser- ies were performed in the granule structure (B). The analyses of the FRAP recovery curves indicated that most of these proteins were immobile. 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 347 543 nm laser line of a CW He–Ne laser through a water immersion objective (C-Apochromat, 40·, 1.2 NA; Carl Ze- iss). Emission signals were detected at > 505 nm for EGFP and > 560 nm for mRFP by single or sequential scanning. FCS setup FCS measurements were carried out with a ConfoCor2 (Carl Zeiss), which consisted of a CW Ar+ laser, a water immersion objective (C-Apochromat, 40·, 1.2 NA; Carl Zeiss), and an avalanche photodiode (SPCM-200-PQ; EG&G, Quebec, Canada). The confocal pinhole diameter was adjusted to 70 lm. Samples were excited with about 10 kWÆcm )2 of laser power at 488 nm, and the fluorescence signal was detected through a dichroic mirror (> 510 nm) and a bandpass filter (515–560 nm). FCS measurement and analysis To remove the chorion, the embryo was peeled with tweezers and put on LAB-TEK chambered coverslips in 1· Yamam- 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  À1=2 where F i and s i are the fraction and diffusion time of compo- nent i, respectively, N is the number of fluorescent molecules in the detection volume element defined by radius w 0 and length 2z 0 , and s is the structure parameter representing the ratio, s = z 0 ⁄ w 0 . FAFs of rhodamine 6G (Rh6G) solution were measured for 30 s three times at 10 s intervals; then the diffusion time (s Rh6G ) and s were obtained by one-component fitting of the measured FAFs. Diffusion constants of samples (D sample ) were calculated from the ratio with the diffusion constant of Rh6G, D Rh6G (2.8 · 10 )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. FRAP curves were created using the following equation: F t ¼ðT 0 À B t Þ=ðT t À B 0 Þ in which F t is the normalized fluorescence at time point t, T 0 and T t represent the fluorescence in the whole cell at time points 0 and t, respectively, and B 0 and B t represent the fluorescence in the bleached region at time points 0 and t. Diffusion constants were determined by classic FRAP analysis [20]. Acknowledgements The authors thank Professor Hiroshi Kimura (Kyoto 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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