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, 151 (6), 1155-68

Nuclear Lamins A and B1: Different Pathways of Assembly During Nuclear Envelope Formation in Living Cells

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Nuclear Lamins A and B1: Different Pathways of Assembly During Nuclear Envelope Formation in Living Cells

R D Moir et al. J Cell Biol.

Abstract

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

Figures

Figure 1
Figure 1
A live PAM cell in metaphase/early anaphase expressing GFP-lamin A observed by phase contrast (a) and fluorescence (b). The GFP-lamin signal fills the cytoplasm, but appears to be excluded from or greatly reduced in the area occupied by the chromosomes. The fluorescence image represents a single confocal section. Bar, 10 μm.
Figure 2
Figure 2
A series of confocal (a–d) and phase contrast (e–h) images of the same PAM cell expressing GFP-lamin B1 as it progresses from early telophase through nuclear assembly in daughter cells. The numbers in the lower right corner refer to the times after the metaphase to anaphase transition. (a and e) Increased fluorescence at the surface of the spindle pole side of the chromosome; (b and f) the same cell taken 2 min later, at which time lamin B1 appears to have completely enclosed the surface of the decondensing chromosomes in late telophase (note the overall reduction in cytoplasmic fluorescence); (c and g) 5 min later, showing that the vast majority of GFP-lamin B fluorescence is located at the surface the daughter cell nuclei, with almost no detectable cytoplasmic fluorescence; (d and h) the daughter cells ∼30 min after the completion of telophase. Note the apparent increase in size of the nucleus. Bar, 10 μm.
Figure 3
Figure 3
Confocal (a–c) and phase contrast (d–f) images of a live BHK-21 cell expressing GFP-lamin B as it progresses from metaphase to telophase. The first image is at metaphase/early anaphase and the subsequent images were taken 8 and 12 min later. Lamin B begins to associate with the surface of chromosomes in the region of the kinetochore facing the spindle poles by mid-late anaphase, 8 min later (b, arrows). This is most obvious in one set of chromosomes and not the other (bottom left) due to the fact that the spindle is slightly tilted. By the time of cleavage furrow initiation in mid-late telophase (4 min later), almost all detectable lamin B fluorescence is seen at the surface of the chromosomes that have reached the spindle poles (c, and f). Bar, 10 μm.
Figure 4
Figure 4
A series of confocal (a–c) and corresponding phase contrast (d–f) images of a live PAM cell expressing GFP-lamin A as it progresses from late telophase through early G1. The numbers in the top left hand corner refer to the times after the metaphase to anaphase transition. (a and b), In late telophase, lamin A remains uniformly distributed throughout the cytoplasm, except for the region containing the chromosomes. 5 min later (b and e), the majority of fluorescence is localized within daughter cell nuclei and the cytoplasmic fluorescence is greatly reduced. The overall pattern of nucleoplasmic fluorescence appears punctate. This pattern appears more obvious 58 min later in both daughter cells (c and f). Bar, 10 μm. Nontransfected PAM cells were fixed and prepared for indirect immunofluorescence with lamin A/C antibody. Early G1 daughter cells were identified by their overall morphology and the presence of a midbody using phase contrast (not shown). The confocal image of G1 cells shows primarily nucleoplasmic staining with relatively little lamin A/C staining in the cytoplasm or in the region of the nuclear lamina (g). GFP-lamin C shows a similar pattern as GFP-lamin A in early G1 (h, compare with c). The differences between B- and A-type lamins in early G1 distribution are seen in doubly transfected cells expressing CFP-laminB1 (blue) and YFP-laminA (green, i). Lamin A is found throughout the nucleoplasm and lamin B1 exclusively at the rim, but there is some overlap at the rim of the two proteins. Bars, 10 μm.
Figure 6
Figure 6
FRAP experiments were carried out on PAM cells expressing GFP-lamin B1 as nuclear assembly begins in late telophase and into G1. A bar-shaped bleach zone was made across the daughter cells and the recovery rate monitored in each of the forming nuclei. For each daughter cell, two bleach zones are introduced in the lamina rim. The recovery rate for each of these zones was estimated and averaged for each cell. For each time point, the prebleach image (a, d, and g), the immediate postbleach image (b, e, and h), and an image taken during the recovery period (c, f, and i) are shown. (a–c) The same nuclei before photobleaching at 10 min after lamin B enclosure (a), immediately after the introduction of a bleach zone (b, arrows), and 10 min after photobleaching (c), at which time the bleached area has almost completely recovered. (d–f) The same daughter cell nuclei immediately before photobleaching at 20 min after lamin B enclosure (d), immediately after photobleaching (e, arrows), and during recovery (f, 65 min after bleaching). During this period, daughter cell nuclei grew and tended to alter their positions slightly. (g–i) The same daughter cell nucleus at 45 min after lamin B enclosure (g), immediately after bleaching (h, arrows), and 180 min later (i, arrows). Only one daughter cell is shown due to the increase in nuclear size. After 180 min (i), the bleach zone shows only partial recovery (arrows). Bars, 10 μm (a–f) and 5 μm (g–i).
Figure 5
Figure 5
PAM cells fixed and prepared for double-label indirect immunofluorescence using antibodies against lamins B or A/C and nucleoporins (Mb414, a–h), NUP153 (i and j), and LAP2β (k and l). A cell in telophase is shown stained with Mb414 (a) and lamin B antibody (b). Lamin B accumulates first at the spindle side of chromosomes before the 414-reactive nucleoporins have begun to accumulate. By the late stages of cytokinesis (early G1), both nucleoporins (c) and lamin B (d) are mainly located in the peripheral region of daughter cell nuclei. In late cytokinesis/early G1, Mb414 stains primarily the peripheral region of daughter cell nuclei (e) and in the same cell the majority of lamin A/C appears distributed throughout the cytoplasm. Later in G1, nucleoporins (g) and lamin A/C (h) are found primarily in the nucleus. Cells in anaphase/telophase show staining at the spindle pole side with NUP153 (i) before lamin B (j). Similarly, LAP2β (k) has completely accumulated around chromatin before lamin B (l). All fluorescence images are confocal. Bars, 10 μm.
Figure 8
Figure 8
(a). A confocal image of live PAM daughter cells in early G1 (20 min after telophase) showing the expression pattern of the GFP–COOH-terminal fragment of lamin B1 that lacks the central rod assembly domain. The mutant lamin is relatively uniform in its distribution throughout the nucleoplasm. (b) In contrast, GFP–wild-type lamin B is mainly located in the lamina region of the daughter cell nuclei at ∼20 min after telophase. Bar, 10 μm.
Figure 7
Figure 7
Confocal images of live PAM cells expressing GFP-lamin B1 before and after extraction with IF buffer. Cells were followed through nuclear assembly and at various time points, the culture medium was removed, and IF buffer was added. An image was collected ∼10 s after extraction. A live cell 20 min after lamin B enclosure (a) and immediately after extraction (b). At this time point, the GFP-lamin B1 remaining after extraction consists of discontinuous spots and lines around the nucleus (arrow). A live cell was also followed for 45 min after enclosure (c), and then extracted with IF buffer (d). In this case, the GFP-lamin B forms an apparently continuous rim around the nucleus after extraction. In both cases (a and b, c and d), there are cytoplasmic lamin foci that have been reported by immunofluorescence in early G1 cells (Goldman et al. 1992; Chaudhary and Courvalin 1993). Bar, 10 μm.
Figure 10
Figure 10
GFP-lamin A and B show intranuclear fluorescence in the majority of interphase PAM cells. (a–e) A live cell expressing GFP-lamin B shows nucleoplasmic (the lamin “veil”) and lamina fluorescence (a) with confocal optics. In b, the cell was photobleached, and after 240 min (c) the bleach zone could still be detected. The t 1/2 for both the nucleoplasm and lamina is similar (∼180 min, see text and Fig. 11). This veil can be seen throughout the nucleoplasm in a confocal through focus series (not shown). (d–f) Live PAM cells expressing GFP-lamin A have a more prominent nucleoplasmic veil. An optical section from an interphase cell expressing GFP-lamin A is shown just before (d), and just after (e) photobleaching. Note that the prominent bleach zone is retained across the entire nucleus, suggesting that this is not an early G1 cell (see text). In f, the same cell was extracted immediately after photobleaching with IF buffer and an image was captured. The veil fluorescence is greatly reduced, but lamina fluorescence appears unaltered. Bars, 10 μm.
Figure 9
Figure 9
Mitotic PAM cells were identified and allowed to proceed until ∼60–90 min after telophase. At this time, both the nucleoplasm and lamina fluoresce. When daughter cell nuclei are photobleached at this time, the recovery in the nucleoplasm is so fast that a bleach zone cannot be detected in a subsequent confocal scan (a). However, the bleached areas in the lamina region recover much more slowly and remain detectable for up to 70 min and later. Only one daughter cell nucleus is shown due to the size of the nucleus. The overall nucleoplasmic fluorescence becomes less intense during this period of observation and the number of nucleoplasmic foci increase. Many of these foci are not continuous with the nuclear surface, as shown through a focus series of images using the confocal microscope (not shown). Bar, 10 μm. A live PAM cell in early G1 expressing GFP-lamin A was photobleached across the entire cell and an image was captured in the subsequent confocal scan (d). As expected for an early G1 cell, the bleach zone is apparent only at the lamina rim (arrows). This same cell was immediately extracted in IF buffer while being viewed on the microscope stage and an image was captured 10-s later (e). The nucleoplasmic fluorescence is almost completely extracted. Bar, 5 μm. FRET analysis was also performed on cells in G1 (e–j). Cells were doubly transfected with pCFP-LB1 and pYFP-LA and examined using a FRET filter setup, as described in Materials and Methods. In cells early in G1 (120–180 min after telophase), the CFP-LB1 (e) was able to activate YFP-LA, resulting in a FRET signal (f), implying these molecules interact. Furthermore, cells very early in G1 (<90 min after telophase), when lamin A is largely nucleoplasmic (YFP-LA; j) also have a FRET signal, indicating an interaction at this time (h and i). Bar, 10 μm.
Figure 11
Figure 11
The fluorescence recovery rates for bleach zones in the lamina rim of interphase cells are shown as an X–Y plot of fluorescence intensity (pixel value) versus pixel position. The average fluorescence intensity (pixel value) of each postbleach image was equalized to that of the prebleach image to compensate for overall fading during image collection. A line along the bleached area of the lamina rim was drawn for each image using the line tool of the Metamorph image analysis program and the profile of fluorescence intensity along the line determined using the line-scan function. The profiles obtained from different time points were plotted and the bleach zone appears as a trough relative to adjacent unbleached areas. Fluorescence recovery was determined by measuring the relative fluorescence recovery in the bleached to the unbleached areas over time. (Top) Recovery for an interphase cell expressing GFP-lamin B1 in interphase. Approximately 50% recovery occurs in 142 min. (Bottom) A cell at interphase expressing GFP-lamin A. This bleach zone has undergone <50% recovery in 3 h.

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References

    1. Benavente R., Krohne G., Franke W.W. Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis . Cell. 1985;41:177–190. - PubMed
    1. Bodoor K., Shaikh S., Salina D., Raharjo W.H., Bastos R., Lohka M., Burke B. Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis. J. Cell Sci. 1999;112:2253–2264. - PubMed
    1. Bridger J.M., Kill I.R., O'Farrell M., Hutchison C.J. Internal lamin structures within G1 nuclei of human dermal fibroblasts. J. Cell Sci. 1993;104:297–306. - PubMed
    1. Broers J.L., Machiels B.M., van Eys G.J., Kuijpers H.J., Manders E.M., van Driel R., Ramaekers F.C. Dynamics of the nuclear lamina as monitored by GFP-tagged A-type lamins. J. Cell Sci. 1999;112:3463–3475. - PubMed
    1. Burke B., Gerace L.A. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986;44:639–652. - PubMed

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