Discontinuous Movement of Mrnp Particles in Nucleoplasmic Regions Devoid of Chromatin

Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin

Jan Peter Siebrassea, Roman Veitha, Akos Dobayb, Heinrich Leonhardtb, Bertil Daneholtc, and Ulrich Kubitschecka,1

aInstitute for Physical and Theoretical Chemistry, Wegelerstrasse 12, Rheinische Friedrich Wilhelms University Bonn, D-53115 Bonn, Germany; bMunich Center for Integrated Protein Science and Department of Biology, Ludwig Maximilians University Munich, D-82152 Planegg-Martinsried, Germany; and cDepartment of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-17177 Stockholm, Sweden

Communicated by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, October 24, 2008 (received for review May 30, 2008) Messenger ribonucleoprotein particles (mRNPs) move randomly they consist of thousands of perfectly aligned chromatids. Tran- within nucleoplasm before they exit from the nucleus. To further scriptionally active regions are expanded, puffed. On chromosome understand mRNP trafficking, we have studied the intranuclear IV there are 2 giant puffs, Balbiani rings (BR) 1 and 2, with movement of a specific mRNP, the BR2 mRNP, in salivary gland cells exceptionally high transcriptional activity. The BR genes are 30–40 in Chironomus tentans. Their polytene nuclei harbor giant chro- kb in size and encode internally repetitive, silk-like proteins. The mosomes separated by vast regions of nucleoplasm, which allows transcripts contain 4 short introns, which are removed predomi- us to study mRNP mobility without interference of chromatin. The nantly cotranscriptionally. The packaging of the large BR tran- particles were fluorescently labeled with microinjected oligonu- scripts with proteins to mRNPs can be visualized in the electron cleotides (DNA or RNA) complementary to BR2 mRNA or with the microscope, the final product being almost spherical particles (2). RNA-binding protein hrp36, the C. tentans homologue of hnRNP The BR particles, Ϸ50 nm in diameter, can be recognized in A1. Using high-speed laser microscopy, we followed the intranu- nucleoplasm and during their translocation through the nuclear clear trajectories of single mRNPs and characterized their motion pore complexes. Approximately a dozen BR-associated RNA- within the nucleoplasm. The Balbiani ring (BR) mRNPs moved binding proteins have been identified, some of which stay associated randomly, but unexpectedly, in a discontinuous manner. When with the transcript when transferred from the gene to the poly- mobile, they diffused with a diffusion coefficient corresponding to somes, e.g., hrp36, whereas others leave the transcript in conjunc- their size. Between mobile phases, the mRNPs were slowed down tion with the passage through the nuclear pore complex (12). By 10-to 250-fold but were never completely immobile. Earlier elec- immunoelectron microscopy of BrUTP-labeled BR particles, it has tron microscopy work has indicated that BR particles can attach to been possible to follow the dispersion of the BR particles from the transcription sites into the vast interchromosomal regions of the cell thin nonchromatin fibers, which are sometimes connected to nucleus (13). It was concluded that the BR particles move randomly discrete fibrogranular clusters. We propose that the observed in nucleoplasm and seem to bind stochastically to the nuclear pore discontinuous movement reflects transient interactions between complexes before exit from the nucleus. freely diffusing BR particles and these submicroscopic structures. The new, powerful light-microscopy technique used to track

single fluorescent molecules or particles with high time resolution CELL BIOLOGY ͉ ͉ ͉ Balbiani ring particles in vivo labeling mRNP trafﬁcking provides detailed information on the molecular dynamics inside ͉ single-molecule ﬂuorescence microscopy single-particle tracking living cells (14). Messenger RNPs can be imaged, because they are large and can be brightly labeled. Two recent studies used synthetic oncomitant with transcription, the growing premessenger genes to produce highly fluorescently labeled mRNA in vivo and CRNA (pre-mRNA) molecules become associated with proteins followed the pathways of these artificial mRNA molecules by to form ribonucleoprotein (RNP) particles (1). The pre-mRNA is single-particle-tracking microscopy (6, 7). processed to mRNA, and the reorganized messenger RNP (mRNP) In the present study, we have analyzed the intranuclear mobility particles are prepared for export (2, 3). After being released from of a single native mRNP species, the BR2 mRNP. Fluorescence the gene, mRNP particles move randomly within the nucleus, labeling was achieved in vivo by microinjection of fluorescent apparently by free diffusion (4). However, the mobility can be oligonucleotides (DNA or RNA), which were complementary to a affected by energy depletion, which suggests that ATP-dependent highly repetitive coding sequence in BR2 mRNA. In addition, we processes also play a role (5). Recently, it became feasible to track examined mRNPs labeled with fluorescent hrp36, a homologue of individual mRNP particles in the nucleoplasm (6, 7). It could then the hnRNP protein A1. Using high-speed laser microscopy, we be confirmed that the particles move by free diffusion, but the followed the intranuclear trajectories of individual mRNPs and diffusion is often spatially constrained. mRNPs travel in interchro- characterized their motion within the nucleoplasm. For reference, matin channels (8, 9), and the movement of mRNPs is probably we determined the intranuclear viscosity by the mobility of inert hindered in tight channels and even stalled in cavities formed by reference particles of known sizes. Finally, a detailed analysis of the chromatin. In the single-particle-tracking experiments, ATP deple- single-trajectory data provided insights into the intranuclear dy- tion showed no effect on the individual, still-mobile mRNPs but namics of mRNPs. further constrained the movement of the particles, probably be- Results cause of a more condensed organization of the chromatin (6, 7). To further study the movement of mRNPs, we have now chosen an In Vivo Fluorescence Labeling of BR2 mRNPs. BR mRNPs are abun- dant in the nuclei of isolated salivary glands, and they can be readily experimental system that allows us to follow the movement of individual mRNPs in large nucleoplasmic regions lacking chroma-

tin, and thus we avoid the complex influence of chromatin. Sur- Author contributions: J.P.S., B.D., and U.K. designed research; J.P.S. and R.V. performed prisingly, we then find that the mRNP particles move in a discon- research; A.D. contributed new analytical tools; J.P.S., R.V., and A.D. analyzed data; and tinuous fashion, suggesting that the particles transiently interact J.P.S., H.L., B.D., and U.K. wrote the paper. with supramolecular assemblies other than chromatin. The authors declare no conﬂict of interest. As the experimental system, we have chosen the salivary gland 1To whom correspondence should be addressed. E-mail: [email protected]. cells of the dipteran Chironomus tentans (2, 10, 11). The nuclei This article contains supporting information online at www.pnas.org/cgi/content/full/ harbor 4 well-demarcated giant chromosomes, which are separated 0810692105/DCSupplemental. by vast regions of nucleoplasm. The chromosomes are polytene, i.e., © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810692105 PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20291–20296 Downloaded by guest on September 25, 2021 A A C

0.05 s 0.1 s 0.15 s 0.2 s 0.25 s B 10.8 7.2 200 nm

[µm] 3.6 y 0

B C 10.8 7.2

[µm] 3.6 y 0 012243648 x [µm] 2.5 D E 5 2.0 4

BR 1.5 3 10 µm 1.0 2

0.5 [µm²/s] MSD 1

C Frequency a.u. Rel. 0.0 0 0 500 1000 0123 Jump Distance [nm] Time [s] 1 min 5 µm 3 min 7 min 10 min Fig. 2. Tracking single BR mRNPs in the nucleus of a living salivary-gland cell. (A) Fig. 1. Electron- and light-microscopic visualization of BR mRNPs. (A) Elec- Five subsequent frames of a movie obtained with the LSM 5 Live. BR2 mRNPs are tron microscopy of BR particles in the nucleoplasm of C. tentans salivary-gland marked by black arrows. Their pathways could be followed by time-lapse imaging cells. Four BR particles are indicated by thin arrows. A minor portion of a BR with 20 Hz. C marks the location of a polytene chromosome. (Scale bar, 2 ␮m.) (B) is demarcated to the left by an dashed line. A segment of an active gene with LSM image of the nuclear envelope marked by Alexa488-labeled Imp ␤1. Selected growing RNP particles has been marked by a thick arrow. (B)AC. tentans trajectories were plotted on the image, which provided a rough structure refer- salivary-gland cell nucleus imaged in an LSM 5 Live after microinjection of ence. (C) Plot of all trajectories obtained from this nucleus. (D) Frequency distri- LgBR-Oligo-Cy5. The white arrows indicate single BR mRNPs. (C) Time course bution of BR2 mRNP jumps performed within 0.05 s. Eq. 3 was used to fit the data of the fluorescence in a living C. tentans salivary-gland cell nucleus after points (bold, solid line). Thin or broken lines indicate distributions for each of the addition of DRB, an inhibitor of RNA polymerase-II-dependent transcription. mobility fractions I–III. (E) Mean-square displacements (MSDs) plotted against the The characteristic labeling of the BR2 by LgBR-Oligo-Cy5 is lost within 10 min. time. The initial slope of the curve was fitted by Eq. 1. This observation indicates that the oligonucleotide probe labeled growing BR2 mRNPs and not the BR2 DNA. Intranuclear Mobility of Single BR mRNPs. Having shown that BR2 mRNPs can be specifically labeled in vivo, we next optimized the recognized in the electron microscope as large, densely stained visualization and tracking of single particles in the nucleoplasm by granules (thin arrows in Fig. 1A) (15). A quantitative analysis using a high-speed laser scanning microscope (LSM). This line- yields a density of approximately 10 BR particles per ␮m3.To scanning LSM yielded high signal-to-noise ratios (SNR) and frame label BR2 mRNPs in vivo, fluorescent DNA oligonucleotides rates. However, because of the high density of labeled mRNPs complementary to a repetitive sequence in BR2 RNA were (approximately 10 per ␮m3), a short photobleaching phase had to microinjected into the nuclei of isolated salivary glands. After be included to singularize particles for tracking. A rectangular injection of minimal amounts of the oligonucleotides, the BR2 region comprising Ϸ1/5 of the nucleus was bleached with high laser transcription site on chromosome IV became clearly visible (Fig. power. Unbleached particles moving into that region could be 1B). This labeling is likely to correspond to nascent mRNA clearly seen. As an alternative to line-scanning LSM, a fast and transcripts, because it disappeared when the transcription inhib- ultrasensitive custom-built single-molecule microscope (SMM) was itor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) was used (16). In these experiments, a circular region within the nucleus coinjected with the oligonucleotides (Fig. 1C). DRB specifically with a diameter of Ϸ15 ␮m was bleached with maximum laser hinders the RNA polymerase II from leaving the promoter but does intensity before movies of unbleached mRNPs tumbling into the not inhibit subsequent transcription steps. Coinjection of the oli- bleached volume were acquired with reduced laser intensity (data gonucleotides with DRB cleared the BR from growing mRNA not shown). transcripts and, thereby, the fluorescence vanished. Hybridization To properly locate the nuclei, we coinjected AlexaFluor488- occurred quickly, and the BRs appeared almost instantaneously labeled importin ␤1 (Imp ␤1) (green) together with red-labeled upon microinjection of the fluorescent probe. Not only nascent oligonucleotides. Imp ␤1 is an import receptor specifically inter- mRNPs were labeled, but also mRNPs that had left the transcrip- acting with nucleoporins that allowed a straightforward localization tion site and were roaming the nucleoplasm (Fig. 1B, white arrows). of the nuclear envelope. However, being living tissues, the glands The labeled particles did not access the polytene chromosomes, were not completely immobile on the coverslip and could move which appeared as dark structures within the nucleus. Experiments slightly during movie acquisition. using Cy5-labeled control oligonucleotides of the same length but The tracking of individual BR2 mRNPs is shown in Fig. 2. Five reverse sequence did not yield any labeling of the transcription site, successive frames from a single LSM movie [supporting informa- and no large particles were detected in the nucleoplasm. tion (SI) Movie S1, which show several single BR2 mRNPs] are In summary, we can use DNA oligonucleotides to specifically displayed in Fig. 2A.Fig.2B gives several examples of trajectories, label both the growing mRNPs in BR2 and the mature BR2 which were deduced by tracking several BR2 mRNPs plotted onto mRNPs released into the nucleoplasmic regions between the a nuclear-envelope reference image, whereas Fig. 2C presents a plot giant chromosomes. of all observed trajectories in a sequence of 23 movies, each 5–8 s

20292 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810692105 Siebrasse et al. Downloaded by guest on September 25, 2021 Table 1. Mobility analysis of single BR2 mRNPs and reference particles

2 2 2 Particle AIII,% DIII, ␮m /s AII,% DII, ␮m /s AI,% DI, ␮m /s n

DNA-labeled BR mRNP 22 Ϯ 9 0.24 Ϯ 0.05 65 Ϯ 7 0.7 Ϯ 0.1 14 Ϯ 4 4.0 Ϯ 1.28 49,547 Qdots 655 40 Ϯ 8 1.43 Ϯ 0.15 60 Ϯ 7 4.17 Ϯ 0.39 3,598 Beads (210 nm) 4 Ϯ 0.9 0.01 Ϯ 0.002 30 Ϯ 11 0.13 Ϯ 0.03 66 Ϯ 11 0.4 Ϯ 0.05 18,743 FITC-dextran,* 500 kDa 100 2.42 Ϯ 0.2

A, relative fraction of total absorbance; n, number of single jumps from frame to frame. *This diffusion constant was determined by a FRAP experiment; see SI Materials.

long. The particle trajectories covered the nucleoplasm quite ho- nucleoplasm, which was examined with fluorescent reference par- mogeneously. Only a small area remained void of mRNPs, and it ticles with diameters similar to those of BR2 mRNPs. Single- was found to be occupied by a polytene chromosome as concluded particle-trajectory analysis yielded the diffusion coefficient D, from the corresponding bright-field image. Occasionally, mRNPs which allowed the calculation of the viscosity by the Stokes– attached to the nuclear envelope, but actual translocations were not Einstein relation, D ϭ kT/6␲␩RS, for a known particle radius RS and recorded, probably because of the chosen time scale or bleaching of temperature T. the particles. First, we analyzed the diffusion of streptavidin-conjugated quan- To describe the mobility of the BR2 particles, we analyzed both tum dots (Qdots 655-Sav) with a Stokes diameter of 26 nm (18). The mean-square displacements (MSDs) and jump-distance distribu- Qdots could be imaged with excellent SNRs, yielding long trajec- tions. From the single-particle trajectories, the MSD can be deter- tories by using the SMM at a frame rate of Ϸ100 Hz (Movie S2 and mined as a function of time, t, yielding the diffusion coefficient, D Fig. 3A). A jump-distance analysis revealed 2 fractions (Fig. 3B), 2 2 (see Materials and Methods). MSD plots, however, produce only with DI ϭ 4.2 Ϯ 0.4 ␮m /s and DII ϭ 1.4 Ϯ 0.2 ␮m /s. Similar averaged diffusion coefficients, which are misleading when differ- experiments were performed with 210-nm microspheres (Fig. 3 C ent types of particles or particles with various mobilities are 2 and D and Movie S3), yielding DI ϭ 0.4 Ϯ 0.05 ␮m/s for the analyzed. Such heterogeneous ensembles are better investigated by majority of all jumps. Finally, we determined the intranuclear analysis of jump-distance distributions, where subpopulations can viscosity by fluorescence recovery after photobleaching (FRAP) be resolved by curve fitting (14). experiments using an FITC-labeled 500-kDa dextran with a Stokes Our jump-distance analysis was based on the trajectories of 4 diameter of Ϸ57 nm. The microinjected probe yielded an intranu- independent experiments (11,256 trajectories) comprising almost clear diffusion constant of D ϭ 2.4 Ϯ 0.2 ␮m2/s (see Fig. S1). All 50,000 single BR2 mRNP jumps between successive frames. The tracer mobility results are summarized in Table 1. analysis of these data revealed that there are at least 3 mobility The apparent intranuclear viscosity was calculated by assuming fractions present (I–III) (Fig. 2D and Table 1). The fastest particles 2 that only the fast fractions represented particles with unrestricted exhibited DI ϭ 4.0 Ϯ 1.3 ␮m /s. Two-thirds of the jumps showed 2 DII ϭ 0.7 Ϯ 0.1 ␮m /s (fraction II), and the slowest fraction (III)

2 CELL BIOLOGY yielded DIII ϭ 0.24 Ϯ 0.05 ␮m /s. In addition to the jump-distance AB11 . 4 2.5 5 analysis, we performed an MSD analysis that yielded an average 4 2 diffusion coefficient DMSD ϭ 0.76 Ϯ 0.03 ␮m /s (Fig. 2E). 2.0 3 2 7.6 1 1.5 [µm²/s] MSD Verification of BR mRNP Identity and Integrity. To further substan- 0 00.100.20

tiate that the BR2 RNA-containing particles are mRNPs, we y [µm] 1.0 Time [s] microinjected fluorescently labeled recombinant hrp36, an hnRNP 3.8 A1-like protein in C. tentans. hrp36 is known to bind in many copies 0.5 Rel. Frequency a.u. Rel. to BR2 RNA and remain associated with the mRNA during 0 0 0 3.8 7.6 11 . 4 0500 1000 transport from gene to polysomes (17). The fluorescent protein x [µm] clearly marked the BR transcription sites on chromosome IV, 3.0 demonstrating the efficient incorporation of the labeled hrp36 into CD3 the BR2 mRNPs (data not shown). Upon injection of very low 2.5 2

amounts of this protein into the nuclei, we detected distinct particles 2.0 1

after a minimum incubation time of 10 min. A jump-distance [µm²/s] MSD 0

1.5 0 1 2 3 analysis of the particle motion yielded a distribution that was similar Time [s] to that of the oligonucleotide-labeled BR2 mRNPs (for details, see 1.0 SI Text). Thus, the labeled particles analyzed are likely to be BR2 10 µm 0.5 mRNPs. Frequency a.u. Rel. The DNA-oligonucleotide-labeled particles displayed a consid- 0 0 500 1000 erably higher mobility than recorded in previous single-particle Jump Distance [nm] experiments (6, 7, 14). To rule out the possibility that the fast motion was caused by fragmentation of the particles by RNase H, Fig. 3. Measurement of the nuclear viscosity with inert reference particles. (A) we performed microinjection experiments using fluorescently la- Sample trajectories of microinjected Qdots in a salivary-gland cell nucleus plotted beled 2Ј-O-methyl-RNA oligonucleotides complementary to BR2 onto the corresponding bright-field image. A part of a polytene chromosome is RNA. Using this approach, we could again identify and specifically visible in the upper image area. (Inset) A typical image of a quantum dot from the track labeled BR2 mRNPs, which yielded results comparable with movie (length, 1 ␮m). (B) Jump-distance distribution of the Qdots655 fitted those from the DNA-oligonucleotide approach (see SI Text). We according to Eq. 3 (solid line) with 2 mobility fractions (dashed lines). (Inset) MSD as a function of time. (C) A movie frame showing microinjected fluorescent conclude that the DNA-oligonucleotide-labeled BR2 mRNPs are microbeads (diameter of 210 nm) within a salivary-gland cell nucleus obtained by not likely to be fragmented by RNase H. the LSM 5 Live at 15 Hz. The overlay of the fluorescence and the bright field image shows the microbeads as bright dots. (D) Jump-distance distribution of the Analysis of the Nuclear Viscosity. An important parameter defining microbeads. The data were fitted (solid line) with Eq. 3 with 2 mobility fractions the intranuclear mRNP mobility is the effective viscosity, ␩,ofthe (dashed lines). (Inset) MSD as a function of time.

Siebrasse et al. PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20293 Downloaded by guest on September 25, 2021 A 2.5 B 0.8 2.0 0.6 1.5 0.4 1.0

0.5 0.2

Rel. Frequency a.u. 0.0 0.0

C 0.7 D0.6 0.6 0.5 0.5 0.4 0.4 0.3 1 µm 0.3 0.2 0.2 0.1 0.1 Fig. 5. Discontinuous movement of BR2 mRNPs. Four examples of long

Rel. Frequency a.u. trajectories, each containing sections corresponding to strongly retarded 0.0 0.0 01000200030000 1000 2000 3000 mobility and others to free mobility. Interval between 2 points, 50 ms. Jump Distance [nm] Jump Distance [nm]

Fig. 4. Jump-distance analysis at different lag times. (A) BR2 mRNP jump- simulations (Fig. S2). We selected all mRNP trajectories containing distance distribution from frame to frame. a.u., Arbitrary units. (B) Jump- Ͼ ϩ jump distances 1,000 nm within 50 ms. Only the mRNPs in the fast distance distribution from frame i to i 10. (C) Jump-distance distribution fraction (4 ␮m2/s) performed such long jumps. In the case of from frame i to i ϩ 20. (D) Jump-distance distribution from frame i to i ϩ 30. A satisfactory ﬁt to the data required 3 mobility components, from which the scenario i, a jump-distance histogram compiled from the selected fraction sizes and the diffusion coefﬁcients were extracted. The broken lines trajectories would recover only a single—the fast—mobility frac- quantify the contributions of the three mobility fractions, whereas the solid tion. However, upon application of this filter to the total experi- line represents their sum. mental trajectory data, we regained at least 2 mobility fractions, thus proving that the trajectories of fast particles also contain retarded sections. This result was supported by visual inspection of mobility. Based on the quantum dots and microspheres, the in- trajectories. In Fig. 5, 4 long trajectories are shown, where segments tranuclear viscosity is 5 centipoise (cP) and 4 cP, respectively. The of high mobility appear to be interrupted by phases of very low FRAP measurements yielded a viscosity of 3 cP. Thus, the effective mobility. Hence, BR mRNPs varied their mobility along the track. intranuclear viscosity is in the 3- to 5-cP range. Discussion BR mRNPs Are Transiently Retarded but Not Completely Immobilized. In this study, we have investigated, at the single-particle level, the Because very slow BR2 mRNPs cannot be detected at short lag intranuclear trafficking of native mRNPs in living tissue by using a times, we also evaluated the jump distances at long lag times. In well-established model system for RNA biogenesis (19). A specific addition to the distances, which were covered after 1 frame or endogenous mRNP particle, BR2 mRNP, was labeled in vivo by 0.05 s (JD1) (Figs. 2D and 4A), we also analyzed those that were fluorescent oligonucleotides complementary to a highly repetitive covered after 10, 20, and 30 frames corresponding to lag times sequence in BR2 RNA. Control experiments with DRB and of 0.5, 1, and 1.5 s, respectively (JD10, JD20, and JD30) (Fig. 4 oligonucleotides with the reversed sequence proved the specificity B–D). Four different mobility fractions could be identified. of our approach. Three of them (I–III) corresponded to the ones discussed above. Two additional fluorescence-labeling strategies, employing ei- In addition, a very slow fraction (IV) was revealed (Table 2). The ther 2Ј-O-methyl-RNA oligonucleotides or an hnRNP protein fast fraction could not be detected after 0.5 s. The observation (hrp36), labeled predominantly the same particles, mostly BR that particles with D ϭ 4 ␮m2/s left the focal plane within 0.5 s mRNPs. Labeling by these quite distinct approaches yielded par- was rationalized by a respective Monte Carlo simulation (see SI ticles with very comparable mobilities, suggesting that the labeling Text). The very slow fraction IV with D ϭϷ0.015 ␮m2/s became did not change the dynamic behavior. Therefore, a mobility analysis noticeable after 0.5 s and was clearly seen after 1 and 1.5 s (Fig. using either approach yields valid results. 4 B–D). Notably, this fraction was not completely immobile but High-speed imaging enabled us to trace the intranuclear trajec- showed a distinct increase in MSDs with time. tories of individual BR2 mRNPs. The Stokes diameter of the BR2 mRNPs was determined to be 36 Ϯ 12 nm, which is in good BR mRNPs Move in a Discontinuous Fashion. The 4 BR2 mRNP agreement with previous electron-microscopy estimations (13), fractions with different mobilities could represent either (i) 4 classes verifying that we indeed visualized single mRNPs and not aggre- of BR2 mRNPs, each with a characteristic constant mobility, or (ii) gates or fragments of mRNPs. The huge amount of data and the a single class of BR2 mRNP particles with varying mobility along considerable length of the trajectories permitted a detailed analysis their trajectories. To decide between the two possibilities, we of the mobility of the BR2 mRNPs. A statistical analysis of all tracks performed a detailed data analysis aided by extensive Monte Carlo with more than 3 positions revealed that the mRNP trajectories did

Table 2. Jump-distance (JD) analysis of single mRNPs for various lag times

2 2 2 2 AIV,% DIV, ␮m /s AIII,% DIII, ␮m /s AII,% DII, ␮m /s AI,% DI, ␮m /s n

JD1* 0.7 Ϯ 0.3 0.015 19 Ϯ 3 0.23 66 Ϯ 2 0.64 Ϯ 0.04 15 Ϯ 3 3.74 Ϯ 1 49,547 JD10 3.5 Ϯ 0.5 0.015 Ϯ 0.003 32 Ϯ 7 0.22 Ϯ 0.03 65 Ϯ 7 0.63 Ϯ 0.04 13,724 JD20 4.5 Ϯ 0.7 0.014 Ϯ 0.003 41 Ϯ 15 0.23 Ϯ 0.04 54 Ϯ 15 0.58 Ϯ 0.09 6,487 JD30 6.6 Ϯ 1 0.015 Ϯ 0.002 19 Ϯ 6 0.13 Ϯ 0.03 74 Ϯ 6 0.43 Ϯ 0.03 3,806

A, relative fraction; n, number of single jumps from frame to frame. *For this ﬁt, a fourth component with DIV and DIII were held ﬁxed.

20294 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810692105 Siebrasse et al. Downloaded by guest on September 25, 2021 not contain more linear, directional sections than trajectories S3). In principle, the transient retardation of the BR2 mRNPs could produced by randomly moving simulated particles (see SI Text). imply that the BR2 particles are temporarily corralled in a small Hence, we could rule out the existence of directed motion for BR2 volume or transiently bound to a large submicroscopic structure. mRNPs, which confirms earlier studies that have shown that Although we cannot exclude the former alternative, we favor the mRNPs move randomly in the nucleus, most likely by Brownian latter because it has been observed by 3D electron tomography that motion. a considerable fraction of the BR mRNPs in the nucleoplasm are We found that the mobility of BR2 particles varied widely. Four associated with thin nonchromatin fibers. These fibers are some- different mobility fractions were identified (I–IV). The fastest BR2 times connected to discrete, complex meshworks of interconnected mRNPs (fraction I) had a diffusion coefficient in the range between fibers and granules, called fibrogranular clusters (25). The fact that ␮ 2 2.7 and 4.7 m /s. The high mobility was not due to a low viscosity the BR particles never completely stop moving would then suggest in the salivary-gland cell nucleoplasm; the effective viscosity was that the thin fibers and the associated structures are mobile and not 3–5 cP, i.e., a viscosity only slightly lower than that in mammalian- likely to be part of a rigid nuclear matrix. Taken together, the cell nuclei (4–8 cP) (20). BR2 mRNPs in fractions II–IV moved morphology and mobility data suggest that BR particles bind considerably slower, with diffusion coefficients of 0.6, 0.2, and 0.015 transiently to thin nonchromatin fibers, which results in a drastic ␮m2/s, respectively. Notably, we found no particles that were completely immobilized. We conclude that BR2 mRNPs in fraction temporary slow-down of the particles. Miralles et al. (25) speculated I are likely to diffuse freely in the nucleoplasm, whereas those in that the submicroscopic structures might modulate the intranuclear fractions II, III, and IV are retarded to an increasing extent. mobility of the BR RNPs. If so, one would expect to find that the in- The high diffusion coefficient of the fast BR2 mRNPs confirms teractions between the BR particles and the thin fibers are nonrandom. earlier results on the most-mobile fraction of RNP particles within In summary, we have demonstrated that native BR2 mRNPs cell nuclei (4). Poly(A)ϩ mRNA was loaded with fluorescent move randomly in the chromatin-free nucleoplasmic regions in the oligo(dT), and the local intranuclear mobility of the resulting C. tentans salivary-gland cell nuclei. However, they travel in a complexes was analyzed by fluorescence correlation spectroscopy discontinuous fashion. When mobile, the particles move as fast as (FCS). Politz et al. (9) speculated that the fast mRNPs rapidly expected from the intranuclear viscosity. We propose that the moved within interchromatin channels. In further studies employ- transient restrictions in mobility are due to interactions between the ing photoactivation or photobleaching techniques (FRAP), BR particles and macromolecular, nonchromatin structures. Such a poly(A)ϩ mRNA was loaded with fluorescent probes, oligo(dT) conclusion is supported by the electron-microscopic observation (21), 2Ј-O-methyl oligoribonucleotides (22), or poly(A)ϩ RNA- that, at a given moment, a considerable number of the BR particles binding proteins (5). These studies reported diffusion coefficients are associated with thin fibers that at least sometimes connect to in the range from 0.03 to 0.7 ␮m2/s only. All numbers represented complex fibrogranular clusters in the nucleoplasm. averages over the complete poly(A)ϩ mRNA population and referred to mRNPs significantly smaller than the especially large Materials and Methods BR2 mRNPs. Despite this fact, the diffusion constants were at least Buffer and Reagents. Transport buffer was used for the dilution of microinjection an order of magnitude smaller than those in our study. It is known, probes [20 mM Hepes/KOH (pH 7.3), 110 mM potassium acetate, 5 mM sodium however, that when using FRAP it is very difficult to correctly acetate, 2 mM magnesium acetate, 1 mM EGTA, and 2 mM DTT]. ZO medium was account for restriction of the movement because of interactions prepared according to ref. 26. 5,6-Dichloro-1-␤-D-ribofuranosylbenzimidazole CELL BIOLOGY (23). In addition, it is not straightforward to analyze mRNP mobility (DRB, Sigma) was dissolved in DMSO and coinjected at a final concentration of 1 by FRAP if small fluorescent ligands are used to tag the particles, ␮M. PBS was prepared from a commercially available stock solution (Biochrom). because the ongoing exchange of bound and free labels leads to Fluorescent DNA and RNA probes used for in vivo hybridization of the BR mRNA complex recovery kinetics (24). These problems are avoided by were from IBA BioTAGnologies. The 30-oligomer BR2.1 DNA probe (ACT TGG CTT using single-particle tracking, where fast, slow, or bound molecules GCT GTG TTT GCT TGG TTT GCT) was either Cy5- or AlexaFluor647-labeled on its Ј are directly discernible. Only 2 recent studies have followed indi- 5 end. An estimate of the number of oligonucleotides that can maximally bind to a single BR2 mRNP is given in SI Text. vidual mRNPs, which were designed to bind multiple fluorophores, As control, Cy5-labeled DNA oligonucleotides of the reversed sequence were either GFP-tagged MS2, a viral RNA binding protein (6), or used. A corresponding 2Ј-O-methyl RNA was labeled by Atto 647N (AttoTec). All molecular beacons (7). In this way, highly fluorescent artificial oligonucleotides were dissolved in water (0.1 nmol/␮l) and diluted 1:10 in trans- mRNPs that could be visualized and traced one at a time were port buffer before microinjection. The human Imp ␤1 was bacterially expressed constructed. In both approaches, diffusion constants that were and labeled with AlexaFluor488. The hrp36 cDNA was kindly provided by N. Visa Ϸ100-fold smaller than the values from the native mRNPs used in (Stockholm University, Stockholm) and subcloned into the pGEX-2T vector (Am- this study were obtained. The reported diffusion coefficients were ersham Pharmacia), thereby introducing an additional cysteine at the hrp36 C conspicuously low, especially considering the shorter length of the terminus. The protein was bacterially expressed as a GST fusion protein, and the mRNAs studied (see discussion of this issue in SI Text). However, GST tag was proteolytically removed. The hrp36 protein was labeled on the these low values were recorded in mammalian nuclei with their introduced cysteine by using maleimide-derivatized dyes. complex chromatin network interfering with the mobility of the mRNPs. We propose that the constraining effect of chromatin is Preparation of Salivary Glands. C. tentans larvae were raised in water-filled, substantial or that the mRNPs in mammalian nuclei also interact aerated plastic dishes and fed with fermented nettle powder (10). Salivary glands with major nonchromosomal structures in the nucleoplasm. were isolated from rapidly growing fourth-instar larvae and kept in PBS during Using single-particle tracking, we detected 4 different mobility preparation. For microinjection and microscopy, the glands were transferred fractions. These fractions could be due either to different classes of onto a polylysine-coated coverslip embedded in a cell culture dish (MatTek). For mobile mRNPs or alternatively to mRNPs altering their mobility incubation under the microscope, either ZO medium or PBS was used. Usually it took 5–10 min to prepare the gland and an additional 5–10 min to microinject 3–6 along the track. The question of which possibility was the case was nuclei. Microinjection was carried out with an Eppendorf injection and micro- answered by a detailed statistical analysis of the trajectory data and manipulation setup, using a holding pressure of 20 hPa and manual injection illustrated by the corresponding Monte Carlo simulations. This procedure. All injection solutions were diluted in transport buffer and centri- approach revealed unambiguously that the mRNPs with high fuged at 22,000 ϫ g for 30 min at 4 °C. mobility also exhibited extended retarded phases along their trajectories (see Fig. 5 and SI Text). Hence, the particles moved in a Fluorescence Microscopy. High-speed confocal imaging was performed with an discontinuous manner but never completely stopped. Phases of very LSM 5 LIVE microscope (Zeiss). Otherwise, an LSM 510-META (Zeiss) was used. For slow diffusive motion occurred all over the nucleoplasm and were single-molecule fluorescence microscopy, a custom-built instrument based on a not confined to regions close to the polytene chromosomes (Fig. commercial inverted microscope was used (16).

Siebrasse et al. PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20295 Downloaded by guest on September 25, 2021 Determination of the Intranuclear Viscosity with Fluorescent Particles. The 3 fluorescent particles were highly diluted in transport buffer (1:1,000 to 1:10,000) Mfj Ϫ 2 Ј͑ ͒ ϭ ͸ r /4Djt and visualized by the LSM5 LIVE or the SMM. We used dark-red fluorescent p r, t dr e rdr, [3] 2Djt polystyrol microspheres with a diameter of 210 nm and streptavidin-coated jϭ1 Quantum dots emitting at 655 nm (Invitrogen). where M is related to the number of jumps considered in the analysis, and f1, f2, and f denote the fractions with diffusion constants D , D , and D , respectively. Data Analysis. Identification and tracking of the single-particle signals were 3 1 2 3 accomplished by using Diatrack 3.01 (Semasopht), a commercial image- processing program for the identification and localization of single-particle Computer Simulation. A computer simulation was designed to simulate diffusing signals and trajectories. All further data processing was performed by using and binding particles in a confined volume representing a nucleus. The particles Origin 7.5 (OriginLab) and ImageJ (27) as described in ref. 14. In the case of moved according to a Gaussian random walk in 3D space. The nucleus was 2 modeled as a sphere of the size of a salivary-gland cell nucleus (radius, 35 ␮m). Brownian motion, the MSDs, ͗r (tc)͘, are linearly related to time and D: Particles hitting the boundaries were reflected, and export was not taken into ͗r2͑t͒͘ ϭ 4Dt. [1] account. For details, see SI Text.

Heterogeneous mobility populations are more appropriately analyzed by a jump- Electron Microscopy. Salivary glands were isolated from fourth-instar larvae. distance analysis. The probability for a particle starting at a specific position to be They were fixed for2hat4°Cin2%glutaraldehyde in 0.05 M sucrose and 0.1 M encountered within a shell of radius r and width dr at time t from that position sodium cacodylate buffer (pH 7.2) and postfixed for1hat4°Cin1%OsO4. The is given as glands were embedded in an agar 100 resin and sectioned in a Reichert Ultracut S ultramicrotome. The ultrathin sections were stained with uranyl acetate and 1 lead citrate and examined in a Philips CM 120 electron microscope. ͑ ͒ ϭ Ϫr2/4Dt ␲ p r, t dr ␲ e 2 rdr [2] 4 Dt SI. Further information is available in Supporting Information including Figs. S4 and S5 and Table S1. when starting at the origin. The probability distribution function can be approx- imated by a frequency distribution by counting the jump distances within respective intervals [r, r ϩ dr] traveled by single particles in a given time. When particles ACKNOWLEDGMENTS. We thank Reiner Peters and Nathalie Fomproix for contributions to the first project phase and Lars Wieslander and Neus Visa for move discontinuously, the jump-distance distributions cannot satisfactorily be stimulating discussions. This work was supported by BioImaging Network Munich fitted by Eq. 2. Different mobility fractions can be quantified by summing up (A.D.), Swedish Research Council and the Knut and Alice Wallenberg Foundation several diffusion terms according to Eq. 2. We mostly used 3 fractions with (B.D.), and Volkswagen Foundation and German Research Foundation (U.K. different diffusion constants, and H.L.).

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