Oligodeoxynucleotides complementary to particular regions of rat 28S rRNA were used as hybridization probes to follow the movement of 28S rRNA within the nucleus of living cells. Target 28S rRNA sequences were selected based on several criteria. First, regions were chosen within expansion sequences, i.e., regions not present in prokaryotic 23S rRNA (Gerbi, 1996
; Dube et al., 1998
), to decrease the likelihood that the hybridized oligos would lie at functional sites of the 60S subunit. The majority of these expansion sequences lie at sites on the 60S subunit that are oriented away from the interface with the 40S subunit (see Beckman et al., 2001
). Second, within these regions, the selected sequences were ones thought to be near the surface of the ribosome, i.e., ones containing nuclease sensitive sites or subject to chemical modification in whole ribosomes, and/or sequences near a binding site for a ribosomal protein known to localize to the ribosomal surface (Han et al., 1994
; Holmberg and Nygård, 1997
; Dube et al., 1998
; Lieberman and Noller, 1998
), but yet not near the face that contacts the small subunit (Holmberg et al., 1994b
; Merryman et al., 1999
). In this way we reasoned that the oligos might find their targets on both incompletely assembled ribosomal precursors as well as on nascent 60S subunits in the nucleolus. Oligos 1 and 2 are targeted to expansion sequences near the binding site for ribosomal protein L25, which is thought to be near the surface of the ribosome. No other proteins have been shown to bind to these particular regions and an antisense oligo targeted to a nearby region was shown to accumulate in nucleoli of mouse cells (Paillasson et al., 1997
). Oligo 3 is targeted to an expansion region near the 5′ end of the 28S rRNA molecule where an insertion has been shown to be viable in yeast (Musters et al., 1989
). Oligo 4 is targeted to a region of the large highly variable expansion sequence D2 (Gerbi, 1996
), where insertion of a marker sequence in Tetrahymena
is known to be viable (Sweeney et al., 1996
). Oligo 5 is targeted to a region in the expansion sequence D12 that is known to be available for chemical modification in mouse ribosomes, indicating that this region may be uncovered near the surface of the ribosome (Holmberg et al., 1994a
). Also, an insert at this site is viable in Tetrahymena
(Sweeney et al., 1996
). , shows the approximate location of these expansion sequences on the surface of the ribosome. shows the five hybridization sites on the folded 28S rRNA molecule.
Figure 1. Targeted hybridization sites on the 60S ribosomal subunit. (A and B) Colored circles show the locations of the expansion sequences for which complementary oligonucleotides probes were designed. (A) Yellow: oligo 1; pink: oligo 2; red: oligo 3. (B) Yellow: (more ...)
Oligodeoxynucleotides complementary to these five regions of 28S rRNA, each 33 nucleotides in length, were synthesized with four, approximately evenly spaced aminohexyl-modified thymidines (see MATERIALS AND METHODS), and these sites were labeled with fluorescein as described (Politz and Singer, 1999
; Politz et al., 2002
). RNA hybrids formed with oligos labeled in this manner are less susceptible to degradation in vivo, perhaps because the evenly spaced aminohexyl arms interfere with RNase H binding (Ueno et al., 1997
; J.C.R. Politz, unpublished results).
Rat L6 myoblasts were allowed to take up a Lipofectamine-bound mixture of all five oligos for 2 h. The medium was changed and after 1 h, cells were examined using digital imaging microscopy on a microscope stage maintained at 37°C (see MATERIALS AND METHODS). Fluorescent signal representing the rRNA oligos was found in the nucleus of transfected cells and additionally was often concentrated in the nucleolus (). Signal was also present in the cytoplasm at lower levels (unpublished data). In parallel experiments it was observed that oligo(dT) or oligo(dA), or oligos containing repeating CTG or CAG sequences did not concentrate in the nucleolus, and in fact, appeared to be excluded from the nucleolus ( and our unpublished results). In standard in situ hybridization experiments with fixed cells, the 28S antisense oligos generated signal in the nucleolus and the cytoplasm as expected (; see also Politz et al., 2002
), whereas only background levels of signal were detected with control oligos ().
Figure 2. Live cell uptake and in situ hybridization of anti-28S rRNA oligos in L6 myoblasts. Cells were allowed to take up Lipofectamine-bound fluorescein-labeled oligos for 2 h as described in MATERIALS AND METHODS. Cells were incubated in fresh medium for 1 (more ...)
We next used an in situ reverse transcription assay (Eberwine et al., 1992
; Politz et al., 1995
) to confirm that the antisense oligos were hybridized to their target 28S rRNA regions in live cells. Cells were again allowed to take up oligo, the medium was changed (see MATERIALS AND METHODS) and the cells were fixed. Hybridized oligo was detected using an in situ reverse transcription reaction. This assay takes advantage of the fact that only hybridized oligo can act as a primer for incorporation of labeled dNTPs by reverse transcriptase; whereas unhybridized oligo cannot (Politz et al., 1995
). shows that signal representing rDNA oligo hybridization was observed in the cytoplasm, and in many cases, also in the nucleolus (red arrows). Only background levels of signal were observed in cells that were not exposed to these oligos (). At higher magnification, the intranucleolar pattern of hybridization appeared generally similar to the fluorescence pattern we observe in live cells, with certain small lobules within the nucleolus showing the most intense signal (unpublished data).
Figure 3. Detection of anti-rRNA oligo hybridization after cellular uptake. Cells were allowed to take up either anti-rRNA oligos (A) or oligo(dT) (C) and were then subjected to in situ reverse transcription to detect sites of oligo hybridization (Politz, 1999 (more ...)
The mixture of all five antisense oligos was next labeled with caged fluorescein (caged-fl; Mitchison et al., 1994
) and introduced into cells as before. The caging groups are two o-
nitrobenzyl moieties covalently linked to fluorescein via photolabile ether bonds. These groups chemically lock the fluorochrome in its nonfluorescent tautomer until photolysis releases the caging groups (Mitchison et al., 1994
; Politz, 1999
; Politz et al., 2003
). The caged-fl rDNA oligos, hybridized to 28S rRNA in the cell, were uncaged in a small 1–2-μm diameter spot using a 360-nm wavelength laser line that was directed through a pinhole and then into the microscope objective. The movement of the resultant fluorescent rRNA was followed as it moved out from the uncaging spot and the 2D signal distribution was recorded every 500 msec using high-speed digital microscopy (Rizzuto et al., 1998
; Politz et al., 1999
). Unless otherwise noted, cells were kept at 37°C throughout the experiment.
Before uncaging, only background levels of fluorescence were detected. When the uncaging beam was directed to nucleoli (which were visualized using phase contrast, , top left, uncaging site circled), the resulting signal was observed to move out in all directions from the nucleolus, and a portion of the signal reached the nuclear periphery by 3.6 s. This pattern of movement, out in all directions from the nucleolar site of uncaging, was consistently observed in >100 cells examined (see also video supplement to ). No evidence of linear paths of signal moving toward a subset of nuclear pores was observed. However, in some cases, a progressive accumulation of signal at a second nucleolus (that was not uncaged) was observed (, bottom panels). We ascertained that uncaged signal was distributed inside the nucleolus and throughout the nucleoplasm in three dimensions by optically sectioning cells after uncaging and subjecting the resulting image stacks to iterative deconvolution analysis (Carrington et al., 1995
). Uncaged signal appeared in all midplanes at all time points, indicating that uncaged signal was distributed throughout the interior of the nucleolus as well as throughout the entire nucleoplasm (unpublished data).
Figure 4. Movement of 60S subunits out from the nucleolus. Cells were allowed to take up a mixture of the five caged-fluorescein labeled anti-rRNA oligos as described in MATERIALS AND METHODS. After a 1-h incubation in fresh medium, oligo in the nucleolus was then (more ...)
In a typical experiment, an average of 63% (range 35–72%) of the signal left the nucleolus () within the 30 s observation period. In contrast, the unhybridized control oligo(dA) left the site much more rapidly; the vast majority was dispersed by 5 s (). The semilog plot in more clearly illustrates the different rates of departure of the control oligo(dA) and the considerably more slowly-moving hybridized rRNA oligos. To analyze the pattern of the rRNA signal movement from the site in more detail, pixel intensities were measured along lines drawn across the nucleus and the nucleolar uncaging site at the various time points (example in ). Signal moved away from the site in a Gaussian distribution, indicative of random movement away from the nucleolus (broad shoulders on blue line in ). A fraction of signal stayed at the nucleolar uncaging site for the duration of the assay period and was often represented by a peak in the center of the plot (, blue line).
Figure 5. Measurement of signal intensity after uncaging. (A) The red curve shows the average percentage of uncaged anti-rRNA oligos remaining at a nucleolar uncaging site compared with the black curve showing the percentage of a nonhybridizing control oligo (oligo(dA); (more ...)
We also measured the movement of 28S rRNA signal within the nucleoplasm (by uncaging away from nucleoli) in similar experiments and again found that the signal moved out from the uncaging site in all directions in a Gaussian profile to fill the nucleoplasm (), with no evidence for directed tracks of signal moving away from the site. We also sometimes observed that a portion of signal uncaged in the nucleoplasm subsequently became concentrated in nucleoli (unpublished data).
To measure the mobility of the 60S subunits as they moved from the nucleolus into the nucleoplasm, we calculated the mean square displacement of signal (as ω2
, the mean square Gaussian width of the signal distribution; see Cardullo et al., 1991
) at different times after uncaging at a nucleolus and plotted the results vs. time. As shown in , ω2
varied linearly with time, as expected for a diffusive process. The slopes of these plots predict that 60S ribosomal subunits move away from the nucleolus with an average apparent diffusion coefficient of 0.31 μm2
/s (SD, ± 0.15 μm2
/s). The nonhybridizing oligo(dA) reached the nuclear membrane too rapidly to allow measurement of a diffusion coefficient using this method; however, we earlier had estimated it to be ~26 μm2
/s using fluorescent recovery after photobleaching (Politz et al., 1998
Figure 6. Mobility and anomalous diffusion characteristics of 60S subunits. The pixel intensity along lines drawn through the center of a nucleolar uncaging site was plotted at different times after uncaging (i.e., ). The mean square displacement (ω (more ...)
Biological processes that involve the consumption of metabolic energy typically display rate differences of 2.0–3.0-fold over a decade of temperature. When the same experiments and analyses as shown in Figures and were repeated at 23°C, rather than at 37°C, approximately the same fraction of signal left the nucleolus during the 30-s assay period, and a similar average apparent diffusion coefficient was observed (0.34 μm2/s, SD, ±0.35 μm2/s). This similar mobility at both 23 and 37°C suggests that the rate of 28S rRNA movement from the nucleolus is not metabolic energy-dependent.
Because the diffusion coefficient measured here was much slower than that predicted for a 60S subunit diffusing in aqueous solution (which we calculate to be ~10 μm2
/s), and because about one third of the uncaged signal did not leave the uncaging site during the assay period, we considered the possibility that the diffusion of the 60S subunits was slowed by collisions and/or retention within nuclear barriers or structures (e.g., chromatin) and therefore was more properly regarded as the phenomenon known as anomalous diffusion. When the log (ω2
) is plotted vs. log dt
, the degree of anomalous diffusion can be determined, and information about the obstacle concentration is also obtainable in some cases (Saxton, 1994
; Platani et al., 2002
). We found that 60S subunit diffusion was indeed anomalous in the nucleoplasm; the log-log plots were linear with a very steep slope (), instead of the zero slope that would be seen with unconstrained diffusion. Anomalous diffusion exponents calculated from these curves were very large (range 4.5–20.7, whereas the exponent in normal diffusion is 2), which indicates that the concentration of diffusion obstacles in the nucleoplasm is very high.