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Obama vs. RyanWhite House spokesman Jay Carney said Tuesday that the plan ...

Obama vs. Ryan

White House spokesman Jay Carney said Tuesday that the plan Obama will outline Wednesday will contain both spending cuts and tax increases. The president is expected to bring back his recommendation, first made in the 2008 campaign, to end Bush-era tax cuts for households earning more than $250,000 a year. Obama also is expected to call for other changes in the tax code, which he contends benefit the rich.

House Budget …

A Game with High Stakes

Andre Mayer called it "playing musical chairs." And Bill Ward said it's a game of "onesies, twosies."

These experts were talking about what is occurring across Western Mass. in terms of the job market.

The good news is that hiring is definitely on the upswing. But it's happening at a snail's pace - one or two jobs at a time, with some employers hiring temporary workers in hopes that the economy will stabilize and they can eventually offer them full-time positions with benefits.

Bright spots exist in the fields of health care, durable-goods manufacturing, and the retail arena, which means there are jobs for people who lack technical skills as well as for those with …

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Literary Listings: Calendar of Local Events

SUNDAY

James Conroyd Martin signs Against of the Crimson Sky: A Novel, 2p.m., Centuries & Sleuths, 7419 W. Madison, Forest Park.

Stuart MacBride signs Dying Light, 2 p.m., Borders, 1500 16thStreet, Oak Brook.

WEDNESDAY

Rick Kogan discuses A Chicago Tavern: A Goat, A Curse, and theAmerican Dream, and Richard Roeper discusses Sox and the City: AFan's Love Affair with the White Sox from the Heartbreak of '67 tothe Wizards of Oz, 6 p.m., Maxim's Nancy Goldberg InternationalCenter, 24 E. Goethe. $20 includes refreshments. Call to reserveseats. (312) 742-1748.

Marisha Pessl signs Special Topics in Calamity Physics, 7:30 p.m.,Women & Children …

Take This, Buckeyes, Huskers And Vols - Autry's the Man

You know all those times you've been in a restaurant or airplaneor movie theater and some adult let his obnoxious kid drive you nuts? Or maybe somebody talked the whole time, or laughed stupidly, ormade you sick with his eating habits?

You always had two thoughts: 1) I'm glad I'm not armed,because there might be hell to pay here; and 2) Some day the tableswill be turned.

Well, today is that day.

College football-wise.

I have my 1995 Heisman Trophy ballot in front of me, No.0424, compliments of the New York Downtown Athletic Club, and as Itype my column, I am laughing.

Let me stop a moment and collect myself.

…

Pomeranz sharp in debut, Rockies beat Reds 4-1

DENVER (AP) — Prized prospect Drew Pomeranz threw five shutout innings in his major league debut and Ty Wigginton homered, helping the Colorado Rockies to a 4-1 win over the Cincinnati Reds on Sunday.

Pomeranz (1-0) was a key piece in the deal that sent ace Ubaldo Jimenez to Cleveland at the trade deadline. He showed why the Rockies coveted him, allowing just two singles.

The 21-year-old lefty threw only 63 pitches before leaving in the fifth. Pomeranz is three weeks removed from an emergency appendectomy.

Edinson Volquez (5-5) was effective in his first start since July 5, not allowing a hit until Wigginton's homer to left in the fourth. He later gave up an RBI single …

AIK coach Stahre moves to Greek club Panionios

Swedish club AIK says coach Mikael Stahre has signed a contract with Greek club Panionios and will leave immediately.

The defending Swedish champions say sports chief Bjorn Wesstrom will succeed Stahre as coach.

Stahre says he wants to try his abilities in an international club and that the decision to leave was …

ComEd ad spokeswoman near the center of power Strobel puts human face on utility's promises

If the name Pam Strobel doesn't ring a bell, this just might:She's the strawberry-blond woman wearing a bomber jacket inCommonwealth Edison television commercials. In one, she's standing infront of an electric substation asking you, the viewer, to take alook at the new ComEd.

"We're not asking you to forget," she says earnestly, tellingviewers and displaying its new slogan that the utility is"reCommitEd" to its customers. Strobel says she can't promise thatthere will never be an electrical outage in Chicago. But in the ads,and to anyone who asks, she vows that it will never be as bad as thatunforgettable summer of 1999, when the Loop went dark, thousands ofhot Chicagoans …

Shields drops 4th straight decision

ST. PETERSBURG, Fla. (AP) — James Shields took the blame for not holding on to a first-inning lead.

Dustin Pedroia, Jacoby Ellsbury and Josh Reddick homered and the Boston Red Sox rallied from an early three-run deficit against the All-Star pitcher to beat the Tampa Bay Rays 9-5 on Saturday.

Shields, coming off a 1-0 complete-game loss to the Yankees in which he allowed the lone run on an errant pickoff throw to third base, gave up four extra-base hits after yielding just five while holding opposing batters to a .178 average over his previous six starts.

"My job was to shut them down and I didn't do my job," Shields said. "When our team scores three runs early in the …

Almagro, Baghdatis, Chela reach quarters in Vienna

VIENNA (AP) — Third-seeded Nicolas Almagro, No. 4 Marcos Baghdatis and No. 8 Juan Ignacio Chela reached the quarterfinals of the Bank Austria Trophy on Wednesday.

Unseeded Michael Berrer of Germany and Andreas Haider-Maurer of Austria also made it to the last eight.

Almagro defeated Colombia's Santiago Giraldo 7-6 (4), 6-7 (3), 6-3 to reach his eighth quarterfinal of the season. The 16th-ranked Spaniard failed to serve out the match at 5-4 in the second set but dominated the decider.

"It was a tough fight and I certainly have to play better in the quarterfinals," Almagro said.

Almagro, seeking his third title of the year, next plays Chela of Argentina, who rallied …

Japan VAM & Poval: Synergy effects by integrating businesses

Japan VAM & Poval Co., Ltd. started on May 1, 2002. Unitika, Ltd. and Shin-Etsu Chemical Co., Ltd. have undertaken the VAM (vinyl acetate monomer) and poval (polyvinyl alcohol) business by joint investments since 1968. Sales divisions of Unitika and ShinEtsu Chemical and factories of VAM and poval were working as four separate companies. On May 1, 2002, they unified and launched a new company.

This move was in order to further raise customer satisfaction and to keep up with the demands of the quick and ever-changing economic environment. Japan VAM & Poval is pursuing to raise output efficiency and the quality of products, to develop a fine technical service, and to perform …

Spain's king to undergo foot surgery

MADRID (AP) — A spokesman says Spain's King Juan Carlos is to undergo minor surgery to repair Achilles tendon damage he suffered since successful knee replacement surgery.

The operation on his left heel will be the 73-year-old head of state's third since May 2010, when he had a benign tumor removed from a lung. In June the king received …

Microenvironment and Effect of Energy Depletion in the Nucleus Analyzed by Mobility of Multiple Oligomeric EGFPs

ABSTRACT

Four different tandem EGFPs were constructed to elucidate the nuclear microenvironment by quantifying its diffusional properties in both aqueous solution and the nuclei of living cells. Diffusion of tandem EGFP was dependent on the length of the protein as a rod-like molecule or molecular ruler in solution. On the other hand, we found two kinds of mobility, fast diffusional mobility and much slower diffusional mobility depending on cellular compartments in living cells. Diffusion in the cytoplasm and the nucleoplasm was mainly measured as fast diffusional mobility. In contrast, diffusion in the nucleolus was complex and mainly much slower diffusional mobility, although both the fast and the slow diffusional mobilities were dependent on the protein length. Interestingly, we found that diffusion in the nucleolus was clearly changed by energy depletion, even though the diffusion in the cytoplasm and the nucleoplasm was not changed. Our results suggest that the nucleolar microenvironment is sensitive to energy depletion and very different from the nucleoplasm.

Abbreviations used: cps, count per second; Cyt, cytoplasm; 2-DG, 2-deoxyglucose; DT, diffusion time; EGFP^sub n^, tandemly linked oligomeric EOFP; FAF, fluorescence autocorrelation function; MR, molecular ruler; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after pholobleach; mRFP, monomeric red fluorescent protein; NP, nucleoplasm; NL, nucleolus; SPT, single panicle tracking; TSA, Trichostatin A.

INTRODUCTION

The cell nucleus contains many proteins that form a multimolecular complex or a material such as chromatin and a nucleolus. Most of the proteins in the nucleus are concerned with molecular processing such as ribosome biogenesis, mRNA synthesis, transcription and molecular transportation to and from the nucleus. For these processes to be accomplished properly, proteins related to each process are expected to act dynamically and precisely in the nucleus. Consequently, the dynamics of various molecules such as RNAs and nuclear proteins in living cells have become a subject of major interest because mobilities of such molecules in the nucleus could provide important information about the molecular functions of the nucleus (1-3). On the other hand, such mobility of functional protein molecules in the nucleus might be mainly affected by the nuclear architecture and microenvironment (1,4) as well as their function because the chromosomes and the nucleoli occupy a large portion of the nuclear space and changes depending on many factors such as gene expression, cell cycle progression, and other metabolic state of the cell. Therefore, for understanding the relation between functional proteins and nuclear microenvironment, it is helpful to analyze mobility of standard protein molecules with well-defined hydrodynamic properties as well as functional nuclear proteins (1,5,6) or labeled macromolecules (7).

In the last few years, many studies based on fluorescence microscopic techniques such as FRAP, single particle tracking (SPT), and fluorescence correlation spectroscopy (FCS) have been carried out for cell biology (8-15). The studies showed that a variety of small fluorescent probes such as BCECF (9), fluorescein-labeled macromolecules (dextran and Ficoll) from 3 to 1000 kD (13), and monomeric EGFP (14), move rapidly in the cytoplasm, whereas labeled linear dsDNA diffuses very slowly and has a size dependence of the diffusion constant (16). The key point of these studies is that the diffusion of small dextrans and Ficolls in the cytoplasm is only restricted mildly whereas that for large macromolecules can be greatly slowed.

On the other hand, a few studies of protein mobility in the cell nucleus have been carried out (10,13,14) with biologically inert protein, even though many studies have been carried out with nuclear proteins (1,3,6). A study based on FRAP and microinjection with diverse sizes of fluorescein-labeled dextrans (13) showed that diffusion in the nucleus was slowed approximately fourfold compared with their diffusion in water. However, more variability in the measured data for the nucleus was found than for cytoplasm. Monomer GFP molecule showed much more complex diffusion in nucleus than in cytoplasm (14). Recent studies of FRAP (1,17) and FCS combined with FRAP experiment ( 18) using living cells have shown that various EGFP-fused nuclear proteins diffuse at different rates depending on their localization and function. Nuclear proteins could interact with target molecules or immobile structures such as chromatin, which slowed down the mobility of the proteins (5,6,19). An FCS experiment with monomeric EGFP showed that diffusion of EGFP, which is presumably inert to other proteins, was restricted depending on the position in the nucleus compared to diffusion in the cytoplasm (14). Furthermore, whether intranuclear mobility of many molecules results from passive diffusion or active transport is still controversial (3,20). The nuclear microenvironment, which may be one of the reasons, has not yet been clearly quantified under various physiological conditions.

FCS has been applied as a powerful technique for assessing biomolecular diffusion and interactions both in aqueous conditions and in living cells with single-molecule sensitivity (21-26). FCS detects fluorescence intensity fluctuations caused by Brownian motion of fluorescent probe molecules in a tiny detection volume (~0.3 fL) generated by confocal illumination. Through time correlation analysis of the fluorescence fluctuations, the diffusion coefficient, the molecular concentration, and the molecular interaction of probe molecules are accessible. Because FCS need only a very small detection volume and has high sensitivity, it will also be useful to measure diffusional mobility of proteins in very small regions of subnuclear microenvironments in living cells. Although FRAP is adequate for measuring the diffusion of fluorescent molecules and possible exchange in target organelles in the living cell (3,7), the measurable minimal fluorescent intensity and diffusional speed range are limited to brighter and slower ranges than those for FCS. Therefore, we can anticipate that FCS will provide complementary information for faster movement at lower expression levels of various functional proteins in the nucleus.

EGFP is a powerful fluorescent bioprobe molecule with a well-known cylindrical structure (27-29). It has recently been used for various cell measurements in fluorescent imaging of cells as well as for analysis of molecular diffusion using FRAP and FCS. To develop a standard and reproducible method for diffusion analysis of proteins, we designed multiple oligomeric EGFPs with different molecular weights, which can be used as molecular rulers (MRs) for quantification of protein mobility in the nucleus. For this purpose, we constructed plasmids with different levels of oligomeric EGFP^sub n^ (EGFP^sub 2^-EGFP^sub 5^, n = 2-5) with molecular weights of 60, 90, 120, and 150 kD, respectively, tandemly linked by a random amino acid linker. Using multiple oligomeric EGFPs and FCS, we determined the diffusion of the proteins in the cytoplasm, nucleoplasm, and nucleoli of living HEK293, HeLa, and COS7 cells. For strict recognition of the two compartments in the nucleus, mRFP-fibriltarin and H2B-mRFP were used as red fluorescent markers for the nucleolus and the nucleoplasm, respectively.

In this study, FCS analysis by a one-component model showed that the diffusional mobility of EGFP^sub n^ in aqueous solution was dependent on the length of EGFP^sub n^ and was well consistent with the diffusion model of a rod-like structure. On the other hand, the diffusion of EGFP^sub n^ in living cells analyzed by a two-component model showed that fast diffusional mobility in the cytoplasm and the nucleoplasm was consistent with the model of a rod-like molecule as shown in aqueous solution. The fast diffusion rates in the cytoplasm and the nucleoplasm were almost the same, and ~3.5-fold slower than in solution, regardless of the size of tandem EGFP^sub n^ and cell type. Mobilities of tandem EGFP^sub n^ found in the nucleoli of HeLa and COS7 cells were fivefold and sevenfold slower than the fast diffusional mobility in the cytoplasm and the nucleoplasm, respectively. Moreover, the much slower diffusional mobility in the nucleolus was also dependent on the length of EGFP^sub n^, demonstrating tandem EGFP molecules were well-defined both in solution and in living cells. Interestingly, the slow diffusion in the nucleolus was related to the energy level of the living cell, because the slow diffusion of EGFPs 'n the nucleolus, but not in the cytoplasm and the nucleoplasm, was further slowed by ATP depletion.

MATERIALS AND METHODS

Plasmid construction of tandem EGFP

Plasmids expressing each tandem EGFP^sub n^ were synthesized with the plasmid expressing EGFP-C1 (Clonteeh, Palo Alto, CA). The EGFP-C1 was excised at the NdeI and the SmaI restriction sites and ligated between the NdeI and Eco47 III restriction sites of another EGFP-CI. The linker between EGFP^sub n^ containing 25 random ammo acid residues (SGLRSRAQASNSAVDGTAGPLPVAT) originated from the remaining bases of the multiple-cloning site. Plasmid constructs of H2B-mRFP and mRFP-fibrillarin were obtained as gifts from Drs. H. Kimura (Kyoto University, Kyoto, Japan (30,31) and T. Saiwaki (Osaka University, Osaka, Japan) (32), respectively. All plasmid constructs for transfection were purified using a plasmid DNA midiprep kit (QIAGEN, Hilden, Germany).

Cell culture and expression of tandem EGFP^sub n^ proteins

For transient expression of tandem EGFP^sub n^, human embryonic kidney 293 (HEK293), HeLa, and COS7 cells were plated at confluence levels of 10-20% on LAB-TEK chambered coverslips with eight wells (Nalge Nunc International, Rochester, NY) for 12 or 24 h before transfection. Cells were transfected with a EGFP^sub n^ vector or cotransfected with a vector of EGFP^sub n^ and H2B-mRFP or mRFP-fibrillarin, and grown in a 5% CO2 humidified atmosphere at 37�C in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 mg/ml streptomycin. Transfection was carried out with FuGENE 6 (Roche Molecular Biochemicals, Mannheim, Germany) or Effectene (QIAGEN) as indicated by the manufacturer. The transfected cells were incubated for 24 or 48 h and washed with Opti-MEM to remove phenol red dye in DMEM, and then the medium was replaced by Opti-MEM before LSM and FCS measurements. Energy depletion was performed by addition of 6 mM 2-deoxyglucose (2-DG, Sigma-Atdrich) and 10 mM sodium azide (NaN^sub 3^, Sigma) to the culture medium (3,20). LSM images were collected for the same cells before and after 2-DG and NaN^sub 3^ treatment.

Western immunoblotting

The immunoblot analysis was performed according to the standard method. Cells expressing tandem EGFP^sub n^ were grown on 10-cm culture plates for 48 h after transfection, the BD Living Colors A.v. peptide antibody (BD Biosciences Cfontech, Mountain View, CA) was used as the primary antibody. Primary antibody-bound protein bands were detected with an alkaline phosphatase-conjugated secondary antibody (mouse anti-rabbit IgG, Chemicon International, Temecula, CA) by BCIP/NBT dye solution (Sigma-Aldrich).

Cell homogenization

After FCS measurements, the cultured cells on a Lab-Tek (Nalge Nunc International) chambered coverslip were collected by centrifugation at 1500 rpm for 5 min and then the pellets of cells were homogenized in 50 �l of buffer (10 mM Hepes pH 7.9 containing 10 mM NaCl, 3 mM MgCl2, 1I mM DTT, 0.4 mM PMSF, and 0.1 mM sodium orthovanadate). Each EGFP^sub n^ protein solution was collected from the supernatant after centrifugalion at 100,000 rpm for 20 min and measured by FCS again.

Live cell imaging

Fluorescence microscopy was performed using an LSM510 inverted confocal laser scanning microscopy (LSM; Carl Zeiss, Jena, Germany). LSM observations were all performed at 25�C. EGFP^sub n^ was excited at 488 nm of a CW Ar^sup +^ laser through a water immersion objective lens (C-Apochromal, 40�, 1.2 NA; Carl Zeiss) with emission detected above 505 nm for single scanning experiments using cells expressing EGFP^sub n^. Monomeric RFP-fibrillarin or H2B-mRFP was imaged using a 543-nm laser light and detection was above 560 nm. The pinhole diameters for confocal imaging were adjusted to 70 �m and 80 �m for EGFP and mRFP, respectively. To avoid bleed-through effects in double-scanning experiments, EGFP and mRFP were scanned independently in a nmltiiracking mode.

FCS measurements and quantitative analysis

RESULTS

LSM observation

Expressed oligomeric EGFP^sub n^ localized in the nucleus

To observe the distribution and localization of monomer EGFP and oligomeric EGFP^sub n^ in HEK293, COS7, and HeLa cells, the cells were transiently transfected with DNA plasmids encoding EGFP^sub n^ or cotransfected with plasmids encoding each EGFP^sub n^ and H2B-mRFP. Cells expressing each oligomeric EGFP were observed at 24-48 h after transfection. Typical LSM images of HeLa cells expressing each EGFP^sub n^ taken at 24 h after transfection are shown in Fig. 1. Monomer EGFP and EGFP^sub 2^ were uniformly distributed through the cytoplasm and nucleus in each cell except in the nucleolus (Fig, 1 A (F) and B (G)). In contrast, EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^ showed different distribution patterns in the cytoplasm and the nucleus. In the case of EGFP^sub 3^, the fluorescent intensity of proteins in the cytoplasm was higher than that in the nucleus, although the difference was not significant (Fig. 1, C and H). For EGFP^sub 4^ and EGFP^sub 5^, the fluorescent intensity in the nucleus was much weaker than that in the cytoplasm (Fig. 1 D (I) and E (J)). However, the fluorescence intensity of EGFP^sub 4^ and EGFP^sub 5^ in the nucleus was sufficient to be detected by LSM measurement (Fig. 1, I and J). The fluorescent intensities in the nucleus for EGFP^sub 2^, EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^ at 48 h were increased compared with these at 24 h. For all oligomeric EGFP^sub n^, there was no speckled or aggregated distribution in the cytoplasm and the nucleopiasm and the fluorescence in the nucleoplasm except in the nucleolus had a uniform pattern (Fig. 1, F-J). This uniform pattern of fluorescence in the nucleoplasm was confirmed by comparing the fluorescence of tandem EGFP with that of H2B-mRFP on HeLa or COS7 cells coexpressing EGFP^sub 3^ and H2B-mRFP or EGFP^sub 5^ and H2B-mRFP, respectively (Fig. S1 in Supplementary Material), because it is known that H2B-GFP show heterogeneous fluorescent pattern in the nucleus depending on the density of chromatin (4,31,38). In the case of HEK293 and COS7 cells transfected with the tandem EGFP^sub n^, the difference of fluorescence intensity between the cytoplasm and nucleus was clearly discriminated from EGFP^sub 3^ regardless of the expression level, and the fluorescent intensity in the nucleus was decreased with the increase in size of tandem GFP (C. Pack and M. Kinjo, unpublished data).

LSM observations of HeLa cell indicated that tandem EGFP bigger than EGFP^sub 3^ (>90 kD) had difficulty localizing in the nucleus. The localization of EGFP^sub n^ in the nucleus was dependent on the size of the EGFP^sub n^ molecule. Although all types of tandem GFP^sub n^ could be localized in the nucleus, there was less tandem EGFP^sub 4^ and EGFP^sub 5^ in the nucleus than monomeric EGFP, EGFP^sub 2^, and EGFP^sub 3^. The small number of EGFP molecules in the nucleus (from 50 to 5 molecules in the detection volume of 0.3 fL) might be sufficient to be detected by FCS measurement even in very weak fluorescent cells. For the weak fluorescent intensity in the nucleus for EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^, we did not need to select weakly expressing cells as explained in Materials and Methods, and could easily perform FCS measurement in the nucleus.

FCS measurement in aqueous solution

Tandem EGFP^sub n^ diffuse in solution like a rod-like molecule

For analysis of the diffusion properties of monomer EGFP and oligomeric EGFP^sub n^ in aqueous solution, cells transfected with the EGFP^sub n^ were homogenized and the proteins from the cell lysate were extracted and measured in aqueous solution. There was no drastic change or burst of average fluorescent intensity during FCS measurement resulting from aggregated EGFP molecules or contaminants from the homogenized cell extracts during the measurement time of 60 s. The FAF of each tandem EGFP^sub n^ was analyzed by a one-component model (Eq. 2, i = 1) and was well fined. Fig. 2 A shows typical FAFs of EGFP^sub n^ obtained from aqueous solution. For comparison of the extents of diffusion speeds, the amplitude of G (τ) (G (0) - 1) was normalized to unity. The autocorrelation functions of EGFP^sub n^ shifted gradually to the right depending on the molecular weight of tandem EGFP^sub n^ (Fig. 2 B). Diffusion times corresponding to the FAFs of EGFP^sub 1~5^ were 86.8 � 3.9 �s, 125.8 � 2.7 �s, 147.4 � 4.8 �s, 185.4 � 5.4 �s, and 200 � 8.5 �s, respectively (Fig. 2 C). These results indicated that the diffusional mobility of EGFP^sub n^ decreased with increasing molecular weight. That there was no degradation of monomeric and tandem EGFP^sub n^ was also confirmed by the Western blotting results (Fig. 2 B), which were well consistent with the expected molecular weight of each oligomeric EGFP^sub n^. Diffusion constants of monomeric and oligomeric EGFP^sub n^ in solution are summarized in Fig. 2 D. The diffusion constant (76 �m^sup 2^s^sup -1^) of monomeric EGFP (263 amino acids, 30 kD) was similar to those (87 �m^sup 2^s^sup -1^) of previous studies (24,39,40) with recombinant GFP (238 amino acids, 27 kD) synthesized by bacterial expression.

Oligomeric EGFP^sub n^ contains a linker of 25 random amino acids connecting monomer EGFP molecules. Consequently, oligomeric EGFP^sub n^ can have different molecular shapes from spherical to linear. Because the linker can change p, the axial ratio of the protein molecule (Eq. 6), the diffusional mobilities of tandem types of EGFP^sub n^ from EGFP^sub 2^ to EGFP^sub 5^ may reflect the diffusional mobility of an ellipsoidal or rod-like molecule. For this case, diffusion times of oligomeric EGFP^sub n^ from EGFP^sub 2^ to EGFP^sub 5^ could be much slower than those of the proteins in spherical shape. Fig, 2 C shows a plot of the measured diffusion time (solid circles) of each EGFP and three plots of predicted diffusion times calculated by diffusion models for the spherical shape and two rod-like shapes with different p-values (Eqs. 5-7). Enhanced EGFP has a well-known cylindrical structure with a diameter of ~3 nm and height of ~4 nm (27). For simplification, monomer EGFP was assumed to be a spherical molecule and then the diffusion time of oligomeric EGFP^sub n^ was calculated as a spherical molecule or rod-like molecule by Eq. 5. The measured diffusion time of monomer EGFP (30 kD) agreed well with the calculated value obtained from Eq. 5 using the empirical diffusion time (21 � 2 �s) and the known molecular weight (0.479 kD) of Rh6G. The dashed line in Fig. 2 C plots the calculated diffusion time of oligomeric EGFP^sub n^ with a spherical shape. The other two lines plot the predicted diffusion times of rod-like oligomeric EGFP^sub n^ assuming that the amino acid linkers have an α-helix (solid line) or a linear structure (dotted line) with lengths of ~3.7 nm and ~9.1 nm, respectively. With this simple assumption, EGFP^sub 1~5^ have longitudinal lengths of 4, 12, 20, 28, and 36 nm, respectively, for an α-helix linker and 4, 17, 30, 43, and 56 nm, respectively, for a linear linker. As shown in Fig. 2 C, the measured diffusion times of oligomeric EGFP^sub n^ (solid circles) are much longer than the calculated diffusion times of the EGFP^sub n^ as a spherical molecule and well agreed with the rod-like model for the α-helix linker, even though the diffusion time of EGFP^sub 5^ was slightly shorter than the calculated value. This indicated that diffusion of monomer and oligomeric EGFP^sub n^ from EGFP^sub 2^ to EGFP^sub 5^ in solution reflected free diffusion of rod-like molecules and depended on the putative length of the oligomeric EGFP. Consequently, we concluded that monomeric and oligomeric EGFP^sub n^ could be used as molecular rulers that change the diffusion time according to their own longitudinal length. This property of tandem EGFP^sub n^ will be useful to analyze mobility of proteins in organelles, particularly in the subnuclear microenvironment.

LSM and FCS measurement in cells

FCS measurements of oligomeric EGFP^sub n^ in vivo were performed using three cell lines, HEK293, COS7, and HeLa. Cells expressing a comparatively low concentration of EGFP^sub n^ under ~20 molecules (<0.1 �M) per detection volume (0.3 fL) were chosen because a dilute concentration of fluorescent molecules is adequate for FCS measurement. Even with this condition, there might be photobleaching effect on FCS measurements. Recently, a method combining FCS with photobleaching analysis was reported for studying intracellular binding and diffusion of functional proteins (41). This study suggested that the method is applicable to analyze mobility of monomer EGFP even in highly fluorescent cells. Nevertheless, it is noted that our study focused on the mobility of freely moving tandem EGFP^sub n^ in the microenvironment containing the detection volume, but not that of immobile tandem EGFP^sub n^, which gives rise to a photobleaching and make FCS analysis more complex. For excluding a possible photobleaching effect, we carefully selected cells with weak fluorescence or without photobleaching during FCS measurement.

All FCS measurements were performed after taking LSM images and multiple positions for FCS measurements in the cytoplasm excepting endoplasmic reticulum and plasma membrane, and multiple positions in the nucleus were chosen in the LSM image of a cell. After FCS measurements, an LSM image was taken again to check whether measured positions of FCS were deviated from the LSM images. In weakly fluorescent cells, it was not easy to discriminate the nucleolus from the nucleoplasm, particularly, in cells expressing EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^, in which most of the proteins were located in the cytoplasm and only a few EGFP molecules were located in the nucleus. Fig. 3 shows typical examples of LSM and FCS measurements for the three cell lines. LSM images for FCS measurement of a HEK cell expressed by EGFP^sub 1^, a COS7 cell by EGFP^sub 4^, and a HeLa cell by EGFP^sub 5^ are shown in Fig. 3, A, C, and E, respectively. On the weakly fluorescent HEK cell expressing EGFP (Fig. 3 A), the boundary between the cytoplasm and nucleus was not clear. On the other hand, the cells expressing EGFP^sub 4^, and EGFP^sub 5^ (Fig. 3, C and E) show a clear contrast of the boundary resulting from the difference of fluorescence intensity between the cytoplasm and nucleus. The boundary between the cytoplasm and nucleus was not clear for weakly fluorescent cells expressing EGFP^sub 1^ and EGFP^sub 2^, regardless of the cell type. However, the boundary was clearly visible with cells expressing EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^, depending on the size of tandem EGFP^sub n^, even though the fluorescence signals of the cells were weak. The clear boundary between the cytoplasm and nucleus for EGFP^sub 3^, EGFP^sub 4^, and EGFP^sub 5^ made it easy to discriminate the two.

FCS analysis in living cells

For all cells expressing EGFP^sub 1^ or tandem EGFP^sub n^, diffusive fluorescent regions in the cytoplasm and multiple positions in the nucleus were measured by FCS. Examples of FAFs of EGFP in HEK, EGFP^sub 4^ in COS7, and EGFP^sub 5^ in HeLa cells are shown in Fig. 3, B, D, and F, respectively. Cross-hairs in the LSM images correspond to the FCS measurement points. In Fig. 3 B, position 1 of FCS measurement point was chosen for measuring cytoplasm, and positions 2 and 3 were presumed to be in the nucleus. In Fig. 3, D and F, position 1 of the cross-hair corresponds to a point in the cytoplasm and positions 2 and 3 to random points in the nucleus. The amplitudes of all FAF (G (0) - 1) were normalized to unity for comparison of the shift of the curve. One of two FAFs obtained from the nucleus showed no or a small difference from that in the cytoplasm (curve 2 in Fig. 3, B, D, and F). Interestingly, other FAFs obtained from the nucleus largely shifted to the right, indicating much slower diffusional mobility (curve 3 in Fig. 3, B, D, and F). This slower diffusion was occasionally found in nuclei of all cells expressing monomeric and oligomeric EGFP^sub n^ regardless of the cell type. This indicated that there were two types of diffusional mobility in the three compartments: the fast-diffusion-mobility (FAF curves 1 and 2 in Fig. 3) in the cytoplasm and the nucleus, and the slow-diffusion-mobility (FAF curve 3 in Fig. 3) in the nucleus (summarized in Table 1). Fluorescent intensity at the point of slower diffusion was weak compared to that at other places in the nucleus. However, we could not specify the precise position of the slow-diffusion-mobility in the cell nucleus because of the very weak fluorescence in the nucleus. Fig. 3 G shows a plot superimposing normalized FAFs of EGFP^sub n^ measured in the nucleus of HeLa cell excluding the slow-diffusion-mobility. The FAF of each oligomeric EGFP in the cell nucleus shifted to the right with the size of tandem EGFP^sub n^. This shift was well consistent with the result in aqueous solution (Fig. 2A). This consistency suggests that the fast-diffusion-mobility of oligomeric EGFP^sub n^ in the cell nucleus might follow the diffusion model of a rod-like molecule.

Analysis of FAF in cells was performed with a twocomponent model ((Eq. 2), i = 1 and 2), a fast diffusing component (first component) and a slower diffusing component (second component), because FAF of each tandem EGFP^sub n^ cannot be fitted by a one-component model, but best fitted by the two-component model. However, some FAFs were best fitted by a one-component model. In this case, we adopted the result of one-component analysis (supplementary Fig. S2). The first component was considered to be a freely diffusing component and the second component was assumed to be a slowly diffusing component (14,24,42). High density of the cellular solutes and some restricted mobility in a cellular microstructure may slow down free diffusion. With conditions of cells having a concentration under 20 EGFP^sub n^ molecules (<0.1 �M) and a comparatively short measurement time under 30 s, the influence of photobleaching on diffusion time, which gives rise to a very long diffusion time and an increase of the fraction (y^sub i^ value in Eq, 2), could be minimized. Photobleaching effects were checked from the time trace of fluorescent intensity for all FCS data (supplementary Fig. S3). Increasing the incubation time after transfection for a few days made the effect of photobleach on FCS measurement much stronger, because the promoter for protein expression is strong and not a controlled one. In practice, photobleaching effect was very small for weakly fluorescent cells at an early stage after expression of tandem GFP (supplementary Fig. S4>. Background fluorescent signals under 2 � 103 cps and 10 � 10^sup 3^ cps were detected in medium and nontransfected HeLa, HEK, and COS7 cells (14). No significant correlation amplitudes were detected in the culture medium. In contrast, very weak correlations with very long diffusion times above 10^sup 5^ �s were sometimes detected in each cell type when FCS measurement was carried out over longer duration over 60 s. This was derived from very slow and large fluctuation of fluorescence but not from photobleaching. To solve the background with very slow fluctuation, we adapted a shorter measurement time as described above. Considering each tandem EGFP has much larger brightness per molecule than that of monomer EGFP (C. Pack and M. Kinjo, unpublished data) and the diffusion time of the proteins was an order of millisecond ranges, the short measurement time of FCS might be enough to obtain a reliable autocorr�lation function.

Two diffusions! mobility in the nucleus

The fast-diffusion-mobility in the cytoplasm and the nucleus

Fig. 4 A shows a plot of the diffusion lime of first component obtained from FAFs representing the fast-diffusion-mobility in the cytoplasm and in the nucleus of HeLa cells (curves 1 and 2 in Fig. 3, B, D, and F). For these FAFs of the fastdiffusion-mobility, >90% of the fraction (y^sub i^ in Eq. 2) was defined as the first component, which represents free diffusion. These results were highly reproducible. As shown in Fig. 4 A (solid circles), the diffusion times of the fastdiffusion-mobility in the nucleus were gradually increased with the increase in the molecular size of tandem EGFP^sub n^. In the cytoplasm (Fig. 4 A, open circles), the diffusion time of the first component for EGFp^sub n^ also increased with size. No significant difference between the first components in the cytoplasm and nucleus was found. Average diffusion times of monomeric and oligomeric EGFP in HEK and COS7 cells also increased with increasing the size both in the cytoplasm and in the nucleus (Fig. 4, B and C). The ratio of the diffusion time of the first component in the cytoplasm and the nucleus of each cell type to that in aqueous solution (DT^sub cell^/DT^sub sol^), which indicates the ratio of viscosity (Eqs, 3 and 4), is shown in inserts in Fig. 4, A, B and C, respectively. Regardless of the cell type, the average ratios of viscosities in the cytoplasm and the nucleus were not significantly different and 3.5-fold higher than that in solution. Moreover, there was no dependency of the viscosity ratio on the size of oligomeric EGFP^sub n^. These results agreed with previous results obtained from microinjected fluorescent macromoleucules and monomeric EGFP (7,13,42).

Using the result that the average viscosity in the cytoplasm and nucleus was 3.5-fold higher than that in solution, the expected diffusion times of tandem EGFP^sub n^ in the cell were calculated. As shown in Fig. 2, the measured diffusion times of first components in living cells were also compared with three calculated diffusion times (Fig. 4, A, B, and C) assuming the shape of oligomeric EGFP to be spherical (dashed lines) or rod-like with the expected linker lengths of 4 nm (solid lines) and 9 nm (dotted lines). Dependency of the diffusion times on the size of oligomeric EGFP both in the cytoplasm and in the nucleus was consistent with that of a rod-like molecule rather than a spherical one (dashed lines). Based on the result that the diffusion properties of rod-like molecules of oligomeric EGFP in the cytoplasm and the nucleus are equivalent and consistent with the result in aqueous solution, the oligomeric EGFP^sub n^ located in the nucleus was not truncated or degraded. Consequently, our results suggested that the diffusion of oligomeric EGFP^sub n^ as a rodlike molecule was well conserved in the cellular circumstance in all of three cell lines.

Other diffusion times (second component) of the FAFs for the fast-diffusion-mobility (curves 1 and 2 in Fig. 3, B, D, and F) were very slow, and ranged from 10^sup 4^ to 10^sup 5^ �-s in the nucleus as well as in the cytoplasm. The range of these long diffusion times was very broad and so it is not clear that the diffusion time of the second component was also dependent on the size of tandem EGFPn. The fraction of the second component (y^sub 2^ in Eq. 2) was very small (< 10%), regardless of the size of tandem EGFP^sub n^. The slow drift of fluorescence could come from cell mobility or very large organelles such as vesicles in cytoplasm (24,42) and such as a compact structure of chromatin in nucleus (4) during FCS measurement. Otherwise very weak photobleaching might be not completely excluded, even though data of photobleached samples were checked and excluded. However, a possibility of trapped diffusion in complex chromatin structures cannot be completely excluded. To analyze an effect of chromatin structures on the very slow diffusion time, we treated cells coexpressing tandem EGFP^sub 3^ and H2B-mRFP or EGFP^sub 5^ and H2B-mRFP, respectively, with Trichostatin A (TSA) (supplementary Fig. S5). It was previously reported that TSA inhibits histone deacetylation and so increases chromatin accessibility of relatively larger dextram (4,43), In LSM observation, no significant changes of fluorescent pattern for tandem EGFP^sub 3^ and EGFP^sub 5^ were found, although that of H2B-mRFP was significantly changed after TSA treatment (supplementary Figs. Sl and S5). This result suggests that tandem EGFP^sub n^ can freely and equally access to all regions of euchromatin and helerochromatin and so no effect of TSA treatment occurred. Moreover, there were no significant changes of diffusion time and fraction for the very slow component in the nucleoplasm after TSA treatment when FCS measurements on euchromatin (dilute H2B-mRFP fluorescent region) and heterochromatin (dense H2B-mRFP fluorescent region) were carried out) (C. Pack and M. Kinjo, unpublished data). Because the fraction of the very slow component was very small (<10%) and the diffusion times were very broad with large standard deviation even before TSA treatment, it is likely that the effect of TSA treatment on mobility of tandem EGFP^sub n^ in the nucleus cannot be detectable in our experimental system. Nevertheless, the result of LSM observation was consistent with the result of FCS measurement. Details and discussion of such very slow diffusion can be omitted in this article because the fraction is small and we focus on well-defined diffusion property of tandem EGFP^sub n^ as molecular ruler.

The slow-diffusion-mobility in the nucleus

On the other hand, the right-shifted FAFs for tandem EGFP^sub n^, which represent the slow diffusion-mobility, found in the nucleus (curves 3, dashed lines in Fig. 3, B, D, and F) showed a different range of diffusion times and a different fraction for the second component compared to those for the fast-diffusion-mobility (curves 1 and 2, solid and dotted lines in Fig. 3, B, D, and F). Obviously, although the diffusion times of the first component for the slow-diffusion-mobtlity in the nucleus were consistent with those for the fast-diffusionmobility in the cytoplasm and in the nucleus (Table 1), the diffusion times of the second component for the slow-diffusion-mobility ranged from 800 to 5000 �s, increasing with the size of tandem EGFP (e.g., curve 3 in Fig. 3, B, D, and F). Moreover, the fraction of the second component for the slow-diffusion-mobility varied from 20 to 100% depending on the cells, and even the measured position in the same nucleus. This observation was very reproducible, and was consistent among the three cell types. Obviously, our results indicated that the protein mobility in the nuclear microenvironment might be separated into two kinds of diffusing species (i.e., the first component of fast-diffusion-mobility and the second component of slow-diffusion-mobility). These two kinds of diffusing species had different ranges of diffusion time (or apparent viscosity) depending on the position inside the nucleus.

The slow-diffusion-mobility of tandem EGFP^sub n^ in the nucleolus

Fluorescent intensity at the position of the slow-diffusionmobility (i.e., the right-shifted FAFs) in the nucleus (position 3 of Fig. 3, A, C, and E) was weak compared to other places inside the nucleus. In addition, the slow-diffusion-mobility was often found in the nucleolus in the cells expressing EGFP^sub 4^ and EGFP^sub 5^ with large and clear nucleoli. The density, the number, and the morphology of the nucleolus changed according to the cell cycle as well as cell type and other cell conditions. Recently, the nucleolus has been detected by fluorescence microscopy in cell lines expressing fluorescent protein-tagged nucleolar proteins such as fibrillarin and B23 (32,44). Fibrillarin is related to various steps of pre-rRNA processing and ribosome assembly and located in the dense fibrillar component (DFC) of the nucleolus during interphase (45). Using a nucleolar protein tagged with different fluorescent proteins will help in discriminating the nucleolar structures from nucleoplasm and tracing the changes of the nuclear structure during the cell cycle or depending on physiological cell conditions.

Fig. 5 shows an LSM image and FCS measurement of a HeLa cell coexpressing EGFP^sub 4^ and mRFP-fibrillarin. The strong red fluorescence in the nucleus (Fig. 5 A) indicates the nucleolus. A weak green fluorescence signal was also detected in the nucleoplasm (Fig. 5 B). This LSM observation for fibrillarin agreed with the previous results (12,32). The shape, the size, and the number of nucleoli were different from cell to cell. Using cotransfected HeLa cells, FCS measurement was carried out for positions of green fluorescent nucleoplasm and the red fluorescent nucleolus with a diameter of over 2 �m in the x-y plane of the LSM image. FAF inside the nucleolus (Fig. 5 D, red line) shifted to the right compared to that in the nucleoplasm (Fig. 5 D, black line), which meant that the diffusion in the nucleolus was much slower than that in the nucleoplasm. The FAFs obtained from nucleolus fit well with the two-component model. Occasionally, some FAFs fit well even in the onecomponent model. The slow-diffusion-mobility in the nucleolus (Fig. 5 D; curve 7) consisted of the first component of 700 �s (40%) and the second component of 3900 �s (60%). In contrast, the fast-diffusion-mobility in the nucleoplasm (Fig. 5 D; curve 2) consisted of the first component of 900 �s (94%) and the second component of 28,000 �s (6%). Diffusion times (1 � 10^sup 3^-4 � 10^sup 3^ �s) of second components for FAFs measured in the nucleolus (Fig. 5 E) were much shorter than those of second components measured in the nucleoplasm and the cytoplasm (ranging from 10^sup 4^ to 10^sup 5^ �s). The time range (Fig. 5 E) was consistent with those obtained from Fig. 3, B, D, and F. In contrast, almost no such diffusional component in the range of 1 � 10^sup 3^-4 � 10^sup 3^ �s was found in places other than the nucleolus. On the other hand, the diffusion time of the first component in the nucleolus was the same as those in the cytoplasm and the nucleoplasm, The fraction of the first component was decreased with the increased fraction of the second component. The diffusion times of second components in the nucleoli increased with the size of EGFP^sub 1^, EGFP^sub 2^, EGFP^sub 3^, and EGFP^sub 4^, even though there was little difference between EGFP^sub 4^ and EGFP^sub 5^ (solid circles in Fig. 5 E). The inset in Fig. 5 E shows the average ratio of the diffusion time of the second component in the nucleolus to the diffusion time of the first component in the nucleoplasm (DT^sub NL^/DT^sub NP^). There was no dependency of the ratio on the size of EGFP^sub n^ and average value of the ratio for all tandem EGFP^sub n^ was ~5.2. The solid line in Fig. 5 E shows the calculated diffusion times of tandem EGFPn as a rod-like molecule with an α-helix linker when the relative viscosity in the nucleolus is fixed by the average ratio of diffusion time (Fig. 5 E, inset). The measured diffusion times of tandem EGFP^sub n^ were consistent with the calculated values. Our results indicated that the slow-diffusion-mobility in the nucleolus also reflected the diffusion of a rod-like molecule rather than a spherical molecule.

Table 1 summarizes the diffusion constants of the fastdiffusion-mobility (the first component with a fraction >90%) found in the cytoplasm and the nucleoplasm, and the diffusion constants of the slow-diffusion-mobility (first and second components) found in the nucleolus. The average values were obtained from living cells only expressing monomer EGFP and tandem EGFP^sub n^ without mRFP-fibrillarin. Diffusion constants of the fast-diffusion-mobility both in the nucleoplasm and in the cytoplasm decreased with the length of tandem EGFP^sub n^ in HeLa, COS7, and HEK cells, even though the diffusion constants of EGFP^sub 4^ and EGFP^sub 5^ in the cytoplasm of HEK cells did not change. Diffusion constants of the first and the second components in the nucleoli of HeLa and COS7 cells also decreased with the length of EGFP^sub n^. There was little difference between diffusion constants in the cytoplasm and the nucleoplasm of HEK293 and HeLa cells. In contrast, diffusion constants in the cytoplasm of COS7 cells were slightly larger than those in the nucleoplasm. Based on these results, it was concluded that the diffusional motion of tandem EGFP^sub n^ in the nucleous as well as in the cytoplasm and the nucleoplasm was well consistent with free diffusion of rod-like molecules, regardless of the cell type. It is emphasized that the microenvironment of the nucleolus as well as the nucleoplasm and the cytoplasm could be quantitatively understood by diffusion analysis of the oligomeric EGFP^sub n^ as molecular rulers (MR). Moreover, our results indicated that the microenvironment and apparent viscosity of the cytoplasm and the nucleoplasm were almost same, even though the constituents of the two compartments were very different.

Compared with those of the first component in the nucleoplasm, the fractions of the second components in the nucleoli were significantly changed from 20 to 100% depending on the nucleolus, even in the same cell (C. Pack and M. Kinjo, unpublished data). Because the length of the z axis (optical axis) of detection volume (<2 �m) was six times longer than the diameter in the x-y plane (<0.2 �m), FCS measurement of a nucleolus with a length in the z axis shorter than 2 �m might contain both the nucleoplasm and the nucleolus. This might affect the variability of the fraction. However, it is also presumed that the diffusion of oligomeric EGFP^sub n^ in the nucleolus has more variability than that found in the cytoplasm and the nucleoplasm, indicating the dynamic change of the nucleolar microenvironment or the complexity of subnucleolar structures such as DFC, fibrillar centers, and the granular region (45,46). More detailed study using two-color 3D imaging combined with FCS measurement is in progress for elucidating the large diffusion changes in the nucleolus according to a long-time scale or the cell cycle of a single cell. Nevertheless, our results showed that the mobility of MR in the nucleolus was dependent onto length of them, but was much slower than those in the cytoplasm and the nucleoplasm. Consequently, it was concluded that the diffusion of protein in the nucleus must be separated into two significant diffusing components, fast-diffusion-mobility in the nucleoplasm and slow-diffusion-mobility in the nucleolus.

Nucleolar microenvlronment Is sensitive to energy depletion

To examine effect of energy depletion on the mobility of oligomeric EGFP in the nuclear microenvironment, the culture medium containing HeLa cells expressing EGFP^sub 4^ or EGFP^sub 5^ was perfused with 2-DG and NaN3 solution (3,20) at 25 or 37�C. LSM and FCS measurements were carried out with HeLa cells expressing EGFP^sub 5^ or coexpressing EGFP^sub 5^ and mRFP-fibrillarin (or H2B-mRFP). For FCS measurement of cells transfected with EGFP^sub 5^, HeLa cells with clear and large nucleoli (>4 �m in diameter) were chosen despite the fluorescence signals of the cytoplasm being a little strong (for example, right upper cell in Fig. 3 E). FCS measurement was carried on the same position of single cells before and after the energy depletion. We confirmed the redistribution of H2B-mRFP and nuclear shrinkage through LSM images of cells coexpressing H2B-mRFP and EGFP^sub 5^ after ATP depletion at room temperature for 30 min (C. Pack and M. Kinjo, unpublished data). This result was consistent with a previous study (20). With cells expressing EGFP^sub 5^, Fig. 6, A-C, show FAFs of EGFP^sub 5^ at the same positions in the cytoplasm, the nucleoplasm, and the nucleolus of a single HeLa cell, respectively, before (dashed black lines) and after the energy depletion (solid red lines). FAFs of EGFP^sub 5^ both in the cytoplasm and in the nucleoplasm were slightly shifted to the right by the energy depletion (Fig. 6, A and B). In contrast, the FAF of EGFP^sub 5^ in the nucleolus was significantly changed in the longer time range as shown in Fig. 6 C. The energy depletion induced a big tail on the FAF, which indicates that a fraction with much slower mobility was newly produced. The diffusion time corresponding to the tail found in the nucleolus was 13,-fold slower than that of the second component found before energy depletion, and the fraction of the new slower component was increased from 0% up to ~32% (Fig. 6 C).

Fig. 6, D-F, shows the average change of the diffusion time and the fraction in each cellular compartment of five HeLa cells expressing EGFP^sub 5^. Averaged diffusion times in the cytoplasm (0.8 � 0.04 ms and 0.85 � 0.04 ms) and the nucleoplasm (0.79 � 0.06 ms and 0.7 � 0.08 ms) before and after ATP depletion, respectively, were not changed (solid bars in Fig. 6, D and E), Instead, the fractions of first components were slightly decreased in the cytoplasm (~1%) and the nucleoplasm (~9%) (Fig. 6, D and E, solid bars in inset). On the other hand, diffusion times of the second component in the nucleolus were increased from 4.6 � 0.8ms to 22.4 � 6.7 ms (Fig. 6 F, open bars), even though the fraction of the second component was decreased from 77 to 47% by the energy depletion (Fig. 6 F, open bars in inset). In addition to the change of the second component in the nucleolus, the diffusion time of the first component in the nucleolus was also increased from 0.58 � 0.1 ms to 1.0 � 0.1 ms (Fig. 6 F, solid bars). This indicated that the microenvronment inside the nucleolus, which was reflected by diffusion of EGFPs molecules, was more sensitive to energy depletion than those of the nucleoplasm and the cytoplasm.

DISCUSSION

Tandemly linked EGFP^sub n^ proteins were constructed for modeling rod-like molecules. The diffusion properties of the proteins were quantitatively dependent on their length. These series of standard proteins allowed us to analyze protein mobility in living cells. LSM observation of HeLa cells expressing monomer EGFP and four different kinds of tandem EGFP^sub n^ showed that the proteins could be distributed to the cell nucleus regardless of their molecular weights. Monomeric EGFP, EGFP^sub 2^, and EGFP^sub 3^ were easily distributed in the nucleus. In contrast, the fluorescent intensities in the nuclei of cells expressing EGFP^sub 4^ and EGFP^sub 5^ were lower than in the cytoplasm, even though they were also located in the cell nuclei. Although the tendency of fluorescence intensity was very much different for EGFP^sub n^ in the nucleus, all tandem proteins were detected by LSM and could also be detected by FCS. Many studies have shown that the transport of inert molecules to the nucleus depends inversely on molecular size with an exclusion limit at ~5-10 nm in diameter or 40-60 kD in molecular weight (47,48). These studies discussed only the exclusion limits of spherical molecules. Our results for tandem EGFP^sub n^ with molecular weights of 60, 90, 120, and 150 kD showed that rod-like proteins could localize to the cell nucleus within 24h after transfection depending on size, even though the mechanism for their transport to the nucleus was not clear.

Western blots of tandem proteins from cell lysates showed that the molecular weights of proteins synthesized in cells were well consistent with those expected from their numbers of amino acids. FCS measurement of monomer and tandem EGFP^sub n^ in aqueous solution showed that their diffusion times also increased with molecular weight. Comparison of the measured diffusion time with the calculated diffusion time according to Perrin's equation (35,36) indicated that the tandemly linked EGFP^sub n^ behaved like rod-like molecules. The fact that diffusion times of tandem series of EGFP are proportional to their lengths in aqueous solution indicates that the proteins could be employed as molecular rulers (MR) in living cells.

Combining a well-defined MR with the high sensitivity of FCS measurement make possible analysis of protein mobility in living cells, in particular in the nucleus. In contrast to the cytoplasm, our results showed that there were two kinds of diffusional mobility in the nucleus, both of which also depended on the length of MR as shown in solution and cytoplasm. One was the fast-diffusion-mobility of tandem EGFP^sub n^ found in the nucleoplasm as well as in the cytoplasm, in which the first component had a fraction above 90%, reflecting the free diffusion of the MR (represented by D of the first components in NP; Table 1). The other was the slow-diffusion-mobility (represented by D of the second components in NL: Table 1) observed in the nucleolus. The second component of the tast-diffusion-mobility in nucleoplasm showed a very slow diffusion (10^sup 4^-10^sup 5^ �s) with fractions under 10%, indicating no length dependency and no significant change by TSA treatment. The first component of the slowdiffusion-mobility (represented by D of the first components in NL, Table 1) in nucleolus was almost equivalent to the first component of the fast-diffusion-mobility in nucleoplasm (represented by D of the first components in NP, Table 1). There was no significant change in the diffusion time and the fraction for the first component of the fast-diffusion-mobility and for the second component of the slow-diffusion-mobility by TSA treatment (C. Pack and M. Kinjo, unpublished data). The result of FCS before and after TSA treatment was well consistent with LSM observation using two-color imaging (supplementary Figs. S1 and S5). Previous studies (4,38,43) using LSM observation of labeled dextran with various sizes showed that a globular protein with molecular weight of 1 MD (an apparent pore size of 14 nm) might be no limitation in access to chromatin. Because molecular weights of tandem EGFP^sub n^ are much smaller than 1 MD, tandem EGFP^sub n^ might freely access the two types of chromatin.

A study of FCS and monomer EGFP using both a two-component model and an anomalous subdiffusion model analysis (14) has shown that the diffusion of EGFP in the nucleus was much more complex than in the cytosol. The study described averaged diffusional mobility of EGFP in the entire nucleus but not in each compartments in the nucleus such as the nucleolus, and suggested that the ratio of diffusion mobilities in cells and in solution was not dependent on the two models used. The fast-diffusion-mobility of tandem GFP^sub n^ in the cytoplasm and the nucleoplasm was dependent on length. The ratio of diffusion time in each compartment to that in solution showed that the apparent viscosities of the cytoplasm and nucleoplasm were identical. In addition, the apparent viscosity in the three cell lines (HeLa, COS7, and HEK293) was found to be ~3.5-fold higher than in aqueous solution. The viscosities in the cytoplasm and the nucleoplasm were well consistent with previous studies using FRAP (9,13) and using FCS (14).

We investigated the protein mobility in the nucleolar microenvironment of living cells in detail. The size and shape of the nucleolus during each phase of the cell cycle are not constant. Moreover, it was not easy to discriminate between the nucleoplasm and nucleolus in the cells weakly expressing the monomer and tandem EGFP^sub n^. We marked the nucleolus with mRFP-tagged fibrillarin to distinguish it from the nucleoplasm. Our observations in the nucleolus (Fig. 5 E and Table 1) indicated that mobility of the inert EGFP and tandem EGFP^sub n^ in the nucleolus was also dependent on the length of the protein, but that the mobility was ~17-fold slower for HeLa and 24-fold slower for COS7 than in aqueous solution. Nevertheless, assuming a random walk model, the result suggested that it would take the tandem proteins just a few seconds to travel a distance of 4 �m, roughly the diameter of a nucleolus. Rapid association or exchange of GFP-fibrillarin (0.046 �m^sup 2^s^sup -1^(12) and GFP-B23 (0.08 �m^sup 2^s^sup -1^) (32) in the nucleolus was observed by FRAP. These results suggested that the nucleolus is not a static protein mass such as aggregates, and that proteins were dynamically exchanged between the nucleoplasm and the nucleolus. EGFP tagged fibrillarin was shown to have diffusion constants of 0.53 �m^sup 2^s^sup -1^ even in the nucleoplasm (12). On the other hand, diffusion of the MR in the nucleolus was much faster than for the nucleolar proteins (Table 1). For instance, the diffusion constants of tandem EGFP^sub 2^ were 14.9 � 0.8 and 3.8 � 0.5 �m^sup 2^s^sup -1^ in the nuceloplasm and the nucleolus of the HeLa cell, respectively, although the molecular weight and shape of EGFP^sub 2^ might be similar to EGFP-tagged fibrillarin (60 kD). Our observations indicated that the architecture of nucleolus was not very tight and some proteins, at least GFP^sub n^, could be almost freely accessible inside of the compartment, because the mobility of GFP^sub n^ was only slowed down about one-fifth compared with the nucleoplasm and the cytoplasm. Consequently, our study of MR mobility in the nucleoplasm and the nucleolus might be very helpful to understand the variability of mobility of microinjected labeled macromolecules in the nucleus (3,13) or the restricted mobility of monomeric EGFP (14) and various nuclear proteins (12,20,32,41,49). In those studies, the complex microenvironment inside of the nucleolus was not considered in detail, even though the mobilities of the nuclear proteins were measured in the nucleolus and the interactions with nucleoli were analyzed.

Recent LSM observation of human U2OS cells expressing yellow fluorescent protein tagged H2B and electron microscopic observation of ATP-depleted cells have shown that the chromatin structure changes with nuclear shrinkage under energy depletion, and suggest that movement of mRNA-protein complexes (mRNPs) is constrained by the structural changes in the nucleus (20). It would be interesting to know whether the redistribution of the chromatin structure by energy depletion also affects other small proteins, and whether the nucleolar microenvironment is also changed by ATP depletion. To determine whether the diffusion of the longest EGFP^sub 5^ in the nucleolar microenvironment was affected by the cellular metabolism, we treated HeLa cells expressing EGFP^sub 5^ with metabolic inhibitors 2-DG and NaN^sub 3^. Interestingly, our results showed that the diffusion of EGFP^sub 5^ in the nucleolus was slowed down by ATP depletion, but that in the cytoplasm and the nucleoplasm it was only slightly changed. The small change of EGFP^sub 5^ mobility (Fig, 6 B) in the nucleoplasm suggested that the microenvironment of nucleoplasm was not so changed. This result indicated that the mobility of proteins smaller than mRNP complex was not sensitive to the structural change in the nucleoplasm (20). Otherwise, the energy depletion would change large nuclear matrix structures (50-52), which affect the much larger molecular size of mRNP (r ~ 133 nm as a circular mRNP with 2.8 kb) rather than that of EGFP^sub 5^ (longitudinal length, ~28 nm). Recent reports have indicated that nuclear diffusion can be limited by a mesoscale viscosity for particles that are larger than 100 nm in diameter (53). In contrast, the change of EGFP^sub 5^ mobility in the nucleolus induced by ATP depletion suggested that the effect of energy depletion on the microenvironment of the nucleolus was bigger than that of the nucleoplasm and cytoplasm, even though the origin of the significant mobility change in the nucleolus was not clear. Nevertheless, our results clearly indicate that the microenvironment of the nucleolus is physiologically very different from that of the nucleoplasm. It is interesting to note that the two motor proteins, nuclear actin and myosin I are related to rDNA and are required for RNA polymerase 1 transcription (54). Such ATP-binding motor proteins can modify the nucleolar microenvironment.

In this study, we have demonstrated that combination of FCS and oligomeric EGFP^sub n^ with different lengths is a novel method to elucidate the nuclear microenvironment of living cells. The microenvironment of the two compartments in the nucleus can now be differentiated and analyzed by using tandem MR, two-color imaging, and FCS. We found that MR EGFP^sub n^, which is presumably inert, could rapidly diffuse inside of the cell nucleolus as well as the nucleoplasm depending only on the length of the protein. Our experimental system can be applied to understanding the mobility of other functional proteins in the nucleolus as well as in the cytoplasm and the nucleoplasm. More importantly, it is also suggested that the microenvironment of the nucleolus is very sensitive to pharmacological energy depletion compared to that of the cytoplasm and the nucleoplasm. Consequently, it is concluded that the physiological state of the nucleolar microenvironment can be understood through mobility analysis of tandem MR in living cells. Combining this method with other fluorescence microscopic methods such as time-lapse microscopy will allow complementary analysis of the nucleolar microenvironment of various cell types and single cells while varying the cell cycle or other physiological conditions such as cell stresses. Effects of GTP depletion or specific inhibitors such as actinomycin D, which primarily affects ribosome biogenesis in the nucleolus through the inhibition of RNA polymerase transcription, will also be important to understand the relations between the nucleolar microenvironment and physiological conditions in detail.

SUPPLEMENTARY MATERIAL

An online supplement to this article can be found by visiling BJ Online at http://www.biophysj.org.

The authors thunk Professor Hiroshi Kimura (Kyoto University, Japan) and Dr. Takuyu Saiwaki (Osaka University, Japan) for providing H2B-mRFP and mRFP-fibrillarin clones, respectively. The authors also thank Dr. Isse Nagao for technical advice for the Western blot experiment.

This research was partly supported by KAKENHI. Orand-in-Aid for Scientific Research (B) by Japan Society for the Promotion of Science (15370062) and Grand-in-Aid for Scientific Research on Priority Areas "Nuclear Dynamics" by MEXT (17050001). C.P. gratefully acknowledges support from 21st Century COE Program for "Advance Life Science on the Base of Bioscience and Nanotechnology" in Hokkaido University, and for "Topological Science and Technology" in Hokkaido University. C.P. is a Postdoctoral Fellow of the JSPS (1705456) in Japan.

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doi: 10.1529/biophysj.105.079467

[Reference]

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[Author Affiliation]

Changi Pack, Kenta Saito, Mamoru Tamura, and Masataka Kinjo

Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan

[Author Affiliation]

Submitted December 9, 2005, and accepted for publication August, 2006.

Address reprint requests to Masalaka Kinjo, Laboratory of Supramolecular Biophysics, R.I.E.S, Hokkaido University, N12W6, Kha-Ku, Sapporo 060-0812, Japan. Tel.: 81-11-7062890; Fax: 81-7064964; E-mail: kinjo@imd.es.hokudai.ac.jp.

Doctor calls for longer turnarounds at World Cup

WELLINGTON, New Zealand (AP) — Scotland team doctor James Robson says the format of the Rugby World Cup may have to be re-examined to avoid short turnarounds between matches which have been criticized by some of the tournament's smaller nations.

Robson said Friday he did not believe matches scheduled as few as four days apart were dangerous but said they were likely to result in below-par performances.

Players and officials from Samoa, Tonga and Canada and Scotland coach Andy Robinson have questioned whether rugby's Tier Two nations should get longer gaps between World Cup matches than their stronger rivals.

"I think to turnaround in international test rugby now, within four days is asking a great deal of the players that we are trying to care for," Robson said.

"So I think from a player welfare point of view, while absolutely cognizant of the commercial needs of world rugby .... . we do have to see if there's some other format for the future where we can have people playing with adequate turnaround time, and therefore performing at their best," Robson said.

Robson said he will bring up the matter at an upcoming International Rugby Board medical conference in London.

"I'm sure we'll have lots to talk about, and one of those things will be how long does it take to actually recover from playing international test match rugby nowadays before you are really fit enough to get back on the pitch and play the next game," he said.

Robson said he had been observing test rugby for 20 years and believed it took at least four days after a major match for players to be able to train "adequately and fully."

"And that's train adequately and fully, not play another game, so I believe it takes at least four days to recover from the rigors of an international test match. I don't think it's dangerous but I think it results in below-par performance."

Robinson has been one of the only coaches of a Tier One team to openly criticize the short turnarounds inflicted on the World Cup's smaller teams.

"I have always banged the drum for the Tier-Two nations because for the game of rugby to truly go global we need 20 teams to be competing to win the World Cup, as you have in soccer," Robinson said after his team's win over Georgia.

"I saw Mike Miller (the IRB chief executive) on our flight today and I said that a four-day turnaround for squads that do not have real depth has to be improved. It is something that should not be allowed to happen if possible. It is better than it was in 2007 and I hope it will be so again in 2015. Teams should have a turnaround of at least five days.

Robinson said Scotland doesn't face the same issues as the smaller teams.

"It was fine for us to play two games in four days because we had the depth of squad, but for the likes of Namibia, facing Fiji and Samoa in four days, it puts them under huge pressure," he said.

Robson said he saw no existing link between short turnarounds and injuries, although he would be eager to study data from the current tournament.

"We see it in most of the teams we play that people are getting better, as in the old adage, we have stronger, fitter, faster people so the collisions are bigger," he said.

"All I can say is people are battered and bruised, they are quite sore and they're quite stiff. It's a hard ask them to do that and to perform at their best and that's really where we want the world state of rugby to be at."

FIFA suspends Belize Football Federation

GENEVA, Switzerland (AP) — FIFA has suspended the membership of the Football Federation of Belize due to 'severe governmental interference.'

The suspensions means that the second leg match for the 2014 World Cup qualifying match between Belize and Montserrat, due to be played Sunday, has been postponed. Belize won 5-2 in the first leg played in Trinidad on Wednesday.

The tie between Belize and Montserrat was the first stage of qualifying for the 2014 World Cup, which will see 203 nations playoff for 31 places at the World Cup finals to be hosted by Brazil.

A statement on FIFA's website said the decision followed the Belize government's informing the FFB on June 8 that it was "not authorized to represent this country in any local or international competition or in any other forum for football on behalf of the Government, People and Nation of Belize."

FIFA said it had no option but to postpone the match after the Belize government said it would not provide the Federation with security services for the visiting team or match officials.

"Under these circumstances, and due to the interference of the government of Belize, FIFA cannot take the responsibility of letting the match take place," FIFA said.

"The match has therefore been postponed to a new date to be confirmed, but no later than July 10, 2011, provided that the situation is back to normal regarding the FFB and the suspension has been lifted by that date."

If the match isn't played by then, FIFA said Belize will be excluded from the 2014 World Cup.

Space-grown soybeans

Soybeans can be grown as a crop in space to provide both food and serve as an atmospheric scrubber for long-term space travel, affirms Tom Corbin, researcher on the mission that put his firm, DuPont, Inc. (Wilmington, DE; www.dupont.com), on the map as the first company ever to complete a major crop growth cycle in space. The 97-d research initiative, which concluded with the return of Space Shuttle Atlantis in October 2002, validated that soybean seeds planted and nurtured by DuPont scientists in the lab had germinated, developed into plants, flowered, and produced new seedpods in space aboard the International Space Station.

During the studies that ensued, the space-grown seeds were manually split - with one part of the seed sown to grow and the other half ground to examine its biological characteristics. DuPont researchers discovered that the space-grown soybeans when compared with their earth-grown counterparts, were similar in physical and biological characteristics, developmental rate, morphology and seed yields. Further, the space-grown seeds were higher in sugar content, but lower in oil and amino acid content, presumably due to the higher CO2 levels on the International Space Station.

Mine ordered to improve sanding after fatality

Federal investigators have directed Consol Energy to properlysand underground tracks at a West Virginia mine where an employeewas killed in a locomotive accident last summer.

The Mine Safety and Health Administration concludes in a reportWednesday there were no contributing violations to the June 5accident.

Consol did not immediately respond to a request for comment.

Veteran 55-year-old miner Gary Hoffman was killed after jumpingor falling from a locomotive at Consol's Robinson Run No. 95 mine inMarion County.

MSHA lists the sanding requirement for part of the mine as thelone enforcement action, saying locomotive operators must be able tostop safely.

Consol operates mines in West Virginia, Pennsylvania, Virginia,Utah, Kentucky and Ohio.

Lifestyle Inflation What it is, what causes it, and what you can do about it.

When did you last upgrade your cellphone? Three months back? Andthe previous one? Another six months? Maybe you're sporting grandertags on your clothes and watches, driving a more expensive car, andspending more on multiplex movies as well. If a growing economy hasput more money into your hands, it has also brought along apropensity to spend more than you require. It's inflation of adifferent kind, lifestyle inflation.

Says Rohit Sarin, Partner, Client Associates: "People's incomeshave gone up. Along with this, attitudes towards spending and savinghave also changed." Another reason for lifestyle inflation is theeasy availability of credit. Credit cards, debit cards and easyfinance schemes have made it possible for the average salaried personto buy items that were earlier beyond reach. So, you'd rather buy aSantro car that costs around Rs 3,60,000 than the ubiquitous Maruti800, which costs around Rs 2,31,000, simply because you can afford topay the EMIs (equated monthly instalments).

While you're welcome to spend your money, it does throw yourfinancial planning into disarray, and this is something financialplanners are struggling with. The way out? Says Ranjeet Mudholkar,CEO, Financial Planning Standards Board, India: "The emphasis onlifestyle costs has to be worked out at an individual level with theplanner. Plans need to be changed to incorporate lifestyle changes."It is therefore important to revisit your financial plans on aregular basis to ensure that you spend sensibly.

But spend and sensibility don't often go together, so here are afew tips. First, try and increase your savings; second, get intoinvesting in assets that are expected to give returns above theinflation rate (such as equity, if you can stomach stock marketchurns, or property). Most important, show some restraint; it's yourmoney, after all.

THE COST OF LIVING IT UP

March 2004 March 2005

Total Annual Income (Rs) 20,00,000 24,00,000

Annual Expenses (Rs)

EMIs (House, Car, etc.) 3,60,000 3,60,000

Cars (Petrol, Maintenance, etc.) 1,50,000 1,80,000

Household Expenses 1,56,000 1,98,000

House Maintenance 25,000 30,000

Domestic Help 48,000 54,000

Kids' Education 1,20,000 1,20,000

Medical Expenses 36,000 50,000

Clothes 1,25,000 2,00,000

Utilities (Phone Bills, Electricity Bills, etc.) 1,10,000 1,25,000

Gadgets (Cellphones, TVs, etc.) 90,000 1,20,000

Socialising/ Entertainment 78,000 1,08,000

Holiday/ Travel 2,00,000 2,50,000

Total Expenses 14,98,000 17,95,000

Increase In Expenses 19.82%

The overall expenses of Rajashekhar (name changed), working as amanager in an MNC, has gone up by nearly 20 per cent, well above thenormal inflation (as measured by the change in wholesale price index)of around 5 per cent. The difference is due to the improvement in hislifestyle (as seen in the increase in spend on clothes, gadgets,entertainment and travel) and can therefore be called lifestyleinflation.