XB-ART-5739J Cell Biol February 17, 2003; 160 (4): 495-504.
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Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle.
Cajal bodies (CBs) are evolutionarily conserved nuclear organelles that contain many factors involved in the transcription and processing of RNA. It has been suggested that macromolecular complexes preassemble or undergo maturation within CBs before they function elsewhere in the nucleus. Most such models of CB function predict a continuous flow of molecules between CBs and the nucleoplasm, but there are few data that directly support this view. We used fluorescence recovery after photobleaching (FRAP) on isolated Xenopus oocyte nuclei to measure the steady-state exchange rate between the nucleoplasm and CBs of three fluorescently tagged molecules: U7 small nuclear RNA, coilin, and TATA-binding protein (TBP). In the nucleoplasm, the apparent diffusion coefficients for the three molecules ranged from 0.26 to 0.40 microm2 s-1. However, in CBs, fluorescence recovery was markedly slower than in the nucleoplasm, and there were at least three kinetic components. The recovery rate within CBs was independent of bleach spot diameter and could not be attributed to high CB viscosity or density. We propose that binding to other molecules and possibly assembly into larger complexes are the rate-limiting steps for FRAP of U7, coilin, and TBP inside CBs.
PubMed ID: 12591912
PMC ID: PMC2173734
Article link: J Cell Biol
Species referenced: Xenopus laevis
Genes referenced: coil herpud1 mtor tbp
Article Images: [+] show captions
|Figure 1. When injected into the cytoplasm, fluorescein-U7 snRNA is assembled into a stable snRNP complex that is targeted to the nucleoplasm and to CBs. (A) Autoradiograph of 32P-labeled snRNAs that were injected into the cytoplasm, recovered the next day from oocyte extract, and immunoprecipitated with anti-Sm mAb Y12. Fluorescein-U7 is immunoprecipitated (lane 2), whereas fluorescein-U7 that contains a mutant Sm-binding site is not (lane 5). Control beads without antibody do not precipitate fluorescein-U7 (lane 3). Pel, material recovered from washed beads; Sup, 10% of supernatant after incubation with beads. (B) Northern blot comparing the total amount of endogenous and injected U7 snRNA in whole GVs. Lane 1, endogenous U7 (end. U7) from 3 control GVs; lane 2, Endogenous and injected U7 (U7 3′ ext.) from 3 GVs isolated 8 h after cytoplasmic injection. Endogenous U6 snRNA serves as a loading control. Arrowhead points to minor breakdown porduct. (C) Top panels; differential interference contrast and fluorescence images of a single brightly fluorescent CB in an oil-isolated GV, 18 h after cytoplasmic injection of fluorescein-U7. Bottom panels; an almost unlabeled CB in a GV that was removed under oil, injected with fluorescein-U7, and held overnight before observation.|
|Figure 2. Xenopus GFP-coilin is efficiently translated by Xenopus oocytes and is targeted to CBs. (A) Western blot of GV proteins from control oocytes (lane 1) or oocytes injected with Xenopus GFP-coilin mRNA (lanes 2 and 3), stained with an antibody against Xenopus coilin (lanes 1 and 2) or against GFP (lane 3). The bottom band is endogenous coilin, the top band is newly translated GFP- coilin. Molecular mass standards in kD. (B) Confocal images of a CB from a control GV (top panels) and from a GV isolated from an oocyte that had been injected with GFP-coilin mRNA (bottom panels), stained with antibodies against GFP (middle panels) or coilin (right panels). In the injected oocyte, GFP-coilin and endogenous coilin are precisely colocalized. CB, Cajal body; N, nucleolus.|
|Figure 3. Human GFP-TBP is efficiently translated by Xenopus oocytes and is targeted to CBs. (A) Western blot of GV proteins from control oocytes (lane 1) or oocytes injected with human GFP-TBP mRNA (lanes 2 and 3), stained with an antibody against human TBP (lanes 1 and 2) or against GFP (lane 3). Both antibodies recognize a band of human GFP-TBP at ∼60 kD. The weak band at ∼40 kD in lanes 1 and 2 may represent endogenous TBP. (B) Confocal images of CBs from a control GV (top panels) and from a GV isolated from an oocyte that had been injected with human GFP-TBP mRNA (bottom panels), stained with antibodies against GFP (middle panels) or human TBP (right panels). In the injected oocyte, GFP-TBP and endogenous TBP are precisely colocalized. CB, Cajal body; N, nucleolus.|
|Figure 4. Oil-isolated GV. (A) Phase contrast (left) and fluorescence image (right) of part of an oil-isolated GV from an oocyte that had been injected with fluorescein-U7. A long exposure (10 s) taken at the oil–GV interface to show the diffuse pool of fluorescein-U7 in the nucleoplasm. (B) Similar images in a region of the GV that contains two CBs. In this short exposure (1 s), the brightly labeled CBs are conspicuous, but the weaker nucleoplasmic label is not evident. B, B-snurposome; CB, Cajal body; NP, nucleoplasm; N, nucleolus.|
|Figure 5. FRAP of fluorescein-U7, GFP-coilin, and GFP-TBP in the GV nucleoplasm. (A) A representative recovery curve of photobleached GFP-coilin in the nucleoplasm of an oil-isolated GV. The curves for fluorescein-U7 and GFP-TBP were similar. (B) Summary of the calculated apparent diffusion coefficient D for fluorescein-U7, GFP-coilin, and GFP-TBP in the nucleoplasm. For each molecule, measurements were made on at least five GVs from at least two animals.|
|Figure 6. The concentration of U7 snRNA in CBs is invariant. CBs in cytological preparations were stained with mAb K121 against the TMG cap found on U7 and other snRNAs. Earlier experiments showed that ∼90% of the TMG staining in CBs is due to U7 snRNA. Total fluorescence of individual CBs from control oocytes was then plotted against their volumes (open circles). The slope of the linear relationship is a measure of TMG concentration. CBs from oocytes that had been injected with U7 snRNA were measured in the same way (filled circles). Despite the consequent increase in nucleoplasmic TMG concentration (see Fig. 7), there was no change in the concentration of TMG in CBs.|
|Figure 7. Exogenous U7 snRNA displaces endogenous U7 from CBs. Northern blot of RNAs isolated from GVs of control oocytes (lanes 1–3) or oocytes injected with a U7 transcript that contained a 6-nucleotide 3′ extension (lanes 4–6). Lanes 1 and 4 (T) contain the total RNA from two unfractionated GVs. Lanes 2 and 5 (P) are from pellets of two fractionated GVs, and lanes 3 and 6 (S) are the corresponding supernatants. In control GVs, the vast majority of endogenous U7 is in the pellet fraction, presumably in CBs (lane 2). In GVs from injected oocytes, some of the extended U7 occurs in the pellet (lane 5) along with a decreased amount of endogenous U7; the rest of the endogenous U7 in such oocytes now appears in the supernatant (lane 6). U6 snRNA, which is predominantly in the supernatant, serves as a loading control.|
|Figure 8. FRAP of fluorescein-U7 in CBs. (A) Selected images from a confocal FRAP series of U7 in a single CB. A diffraction-limited spot was bleached inside the CB, and recovery of fluorescence was recorded as a function of time in seconds. Bar, 5 μm. (B) Plot of means and SDs of U7 data from 15 experiments of the sort shown in A. In each experiment, the average intensity of the bleach spot was measured as a function of time. Intensities were normalized to the prebleach value and corrected for minor bleaching during imaging. The curve for GFP-coilin was almost identical to that of U7. For GFP-TBP, the final phase of recovery was somewhat slower.|
|Figure 9. FRAP of fluorescein-UTP and Alexa 546–U7 in CBs and nucleoplasm. An oocyte was coinjected in the cytoplasm with fluorescein-UTP and Alexa 546–U7 snRNA. After overnight incubation, the GV was isolated in oil and squashed under a coverslip. Fluorescein-UTP was neither concentrated in nor excluded from CBs relative to the nucleoplasm (A), whereas Alexa 546–U7 accumulated strongly in the same CB (B). Spots were bleached in the nucleoplasm and in the CB (white dots in the prebleach images). Recovery in the fluorescein-UTP channel (A) was too fast to record in both the nucleoplasm and CB after bleaching. Therefore, the postbleach image is uniform in intensity (except for two B-snurposomes; one on the surface and one inside the CB, which bind fluorescein-UTP weakly). Recovery of the bleach spot (arrowhead) inside the CB is very slow in the Alexa 546–U7 channel (B). The postbleach images represent a single confocal scan and are grainier than the prebleach images, which are frame-averaged.|
|Figure 10. Kinetic models of CB function. (A) A sequential model in which molecules pass from one state(s) to the next in a linear, irreversible manner before exiting into the nucleoplasm. This model is incompatible with the kinetics of our FRAP data. (B) A more complex model in which the initial binding event (s1) is reversible and fast, and the slowest step (s3) corresponds to a reservoir that is kinetically isolated from the state that exits the CB (s2). This model is compatible with our FRAP data.|
References [+] :
Andrade, Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. 1991, Pubmed