Diffusion-based transport of nascent ribosomes in the nucleus - PubMed (original) (raw)
Diffusion-based transport of nascent ribosomes in the nucleus
Joan C Ritland Politz et al. Mol Biol Cell. 2003 Dec.
Abstract
Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.
Figures
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: oligo 4; orange: oligo 5 (see MATERIALS AND METHODS for sequences of each oligo). The cryo-EM maps of the S. cerevisiae 80S ribosome are taken from Spahn et al. (2001) and reproduced with permission of CELL Press (Cambridge, MA). (C) 28S secondary structure map (yeast) showing regions targeted for oligo hybridization in red.
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 h and then examined using digital imaging microscopy. (A) Phase and fluorescent images of live cell nucleus after uptake of the five anti-28S rRNA oligos. Cytoplasmic signal is not visible because the image is scaled so that nuclear detail can be seen. (The bright nuclear spots are not SC35 rich speckles [our unpublished results] and are too plentiful to be Cajal bodies; similar bright spots are observed after oligo(dT) uptake.) (B) Phase and fluorescent images of a live cell nucleus that has taken up, as a control oligo, a 33mer repeat of CAG labeled in the same way as the oligos in A. (C) Phase image and in situ hybridization of anti-rRNA oligos to fixed cells. (D) Phase image and in situ hybridization of CAG control oligos (see B) to fixed cells.
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; Politz et al., 1999; see also MATERIALS AND METHODS). This assay exploits the fact that only hybridized oligo will prime reverse transcription and incorporation of labeled nucleotides; unhybridized oligo will not. Dark signal represents sites at which incorporated label, and thus hybridized oligo, is detected. Red arrows in A point to label present in nucleoli. (B) Results when the cells were not exposed to oligo.
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 uncaged and the movement of the signal was tracked over time using high-speed digital imaging techniques (see MATERIALS AND METHODS). The signal moved out in all directions from the nucleolus (top panel) and in some cases the uncaged nucleolar signal moved to the other nucleolus in the nucleus (bottom panel). See also supplemental video.
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); see Politz et al., 1999) remaining after uncaging in the nucleoplasm (control oligos do not localize to the nucleolus). The red curve here represents the average of 11 cells uncaged in one experiment, and similar results were obtained in four other experiments. (B) Same data as in A on a semilog plot. (C) Pixel intensities measured along a line drawn across the nucleus and the nucleolar uncaging site showing a Gaussian distribution of signal intensity at 110 msec (purple line) and 30 s (blue line) after uncaging. (D) Line intensity plot through center of an uncaged spot in nucleoplasm at 110 msec (purple) and 30 s (blue) after uncaging. The spatial distribution of signal after nucleoplasmic uncaging was similar at both 37 and 23°C.
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., Figure 5C). The mean square displacement (ω2) over time was then calculated as the average of the radius of the uncaged signal distribution measured at the points at which the intensity had fallen to e_–2 of the maximum intensity within the uncaged spot (because the signal is distributed in a Gaussian). (A) Values from a typical experiment plotted vs. time. In this experiment, the apparent average diffusion coefficient estimated from the slope of the line (slope = 8_D; see Cardullo et al., 1991) was 0.44 μm2/s. (B) Log-log plot of ω2/dt vs. dt shows anomalous diffusion. Values from the experiment shown in A fall on a straight line with slope of 2/dw – 1 (see Saxton, 1994), where the anomalous diffusion exponent dw equals 4.5. The red lines show a linear least-squares fit to each plot.
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