The movement of coiled bodies visualized in living plant cells by the green fluorescent protein

Abstract

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body.

Figure 1

(A) Single confocal optical section of BY-2 cells expressing U2B"-GFP, double labeled with GFP (left panel) and autoantibody against p80 coilin (right panel). Three nuclei are shown, and the bright GFP spots colocalize with bright foci of anti-coilin labeling. There is some labeling of the cytoplasm by anti-p80 coilin. (B) Single confocal optical section of BY-2 cells expressing U2B"-GFP, double labeled with GFP (left panel) and 4G3 antibody (right panel). Three nuclei are shown. Most coiled bodies are in the nucleoplasm, but occasionally are seen in the nucleolus (arrows). All coiled bodies that contain U2B" also express the U2B"–GFP fusion. Bars, 5 μm.

Figure 2

Confocal fluorescence microscopy of U2B"-GFP in a living BY-2 cell. (A) Single confocal optical sections of a BY-2 cell nucleus are shown, with 2.6 μm between consecutive sections, followed by a projection of the 3-D data stack. Coiled bodies can be found in the nucleoplasm and nucleolus, but most frequently just inside the nucleolar periphery. (B) Stereo projection of the confocal series shown in panel A. Bar, 5 μm.

Figure 3

Time-lapse confocal microscopy of U2B"-GFP in living BY-2 cells. Projections of three or four confocal optical sections are shown at each time point. Coiled body marked with an arrow demonstrates that coiled bodies can move around in the nucleoplasm and nucleolus, and often move along the periphery of the nucleolus. Other, unmarked coiled bodies show smaller movements. Bar, 5 μm.

Figure 4

Time-lapse confocal microscopy of U2B"-GFP in living BY-2 cells. Projections of series of confocal optical sections are shown at each time point. Coiled body marked with an arrow moves from the periphery of the nucleoplasm to the periphery of the nucleolus, after which it moves along the periphery of the nucleolus and finally fuses with another coiled body. Other coiled bodies also show movements. Bar, 5 μm.

Figure 5

Time-lapse confocal microscopy of U2B"-GFP in living BY-2 cells. Projections of series of confocal optical sections are shown at each time point. Late telophase/early G₁ cells have many small coiled bodies. As the cells progress through the cell cycle, the nuclei enlarge and the number of coiled bodies decreases. Concomitantly, the size of the coiled bodies increases. Bar, 10 μm.

Figure 6

Time-lapse confocal microscopy of U2B"-GFP in stable Arabidopsis transformants. Projections of series of confocal optical sections through the root epidermis are shown at each time point. Many coiled bodies are clearly mobile. Several coiled bodies coalesce (arrows). Two cells underwent division during this sequence, and the newly divided cells decreased their coiled body numbers quickly (asterisk). Bar, 10 μm.

Figure 7

Heat shock on U2B"-GFP–expressing BY-2 cells. Projections of series of confocal optical sections are shown at each time point. (A) Cells before heat shock. (B) After 1 h heat shock, at 42°C, all coiled bodies have disappeared. (C) 19.5 h after heat shock, coiled bodies have reformed. (D) A second heat shock, at 42°C, for 1 h, results in the disappearance of all coiled bodies. (E) 2.5 h after heat shock, coiled bodies are already present. Several coiled bodies reappear near places where they were before heat shock (arrows). Bar, 5 μm.

Figure 8

Actinomycin D treatment of U2B"-GFP–expressing BY-2 cells. Projections of series of confocal optical sections are shown at each time point. After addition of 2 mM actinomycin D, at time point 0 h, U2B"-GFP forms elongated structures around the nucleolus (arrow). These elongated structures change their relative positions around the nucleolus and can increase or decrease in size. Bar, 5 μm.

Figure 9

Okadaic acid treatment of U2B"-GFP–expressing BY-2 cells. A single confocal section is shown. After 19 h treatment with 100 nM okadaic acid, coiled bodies are mainly found in the nucleolus in BY-2 cells. Bar, 5 μm.

Figure 10

Stable U2B"::GFP expression in Arabidopsis roots. Projections of series of confocal sections through the root meristem are shown. (A) The full-length U2B"::GFP fusion shows expression in the nucleoplasm and in the coiled bodies (bright spots) in all root nuclei in the epidermis. The nucleolus is largely devoid of labeling (dark centers). Root cap nuclei are labeled very brightly (small nuclei). (B and C) Deletion constructs U2B"del1::GFP and U2B"del2::GFP label the nucleoplasm and the coiled bodies. In addition, cytoplasmic strands emerging from the nucleus and plastid-like structures are clearly labeled in the root meristem of U2B"del1::GFP transformants. (D) Fusion of the U2B" putative NLS to GFP results in labeling of the cytoplasm and somewhat brighter labeling of the nucleoplasm. There is no labeling of coiled bodies. Bar, 20 μm.