Localization of Integral Proteins of the NE in Relation to Bulk ER Membranes during Mitosis by Confocal Microscopy
We have investigated the fate of integral membrane proteins of the NE during mitosis by immunofluorescence staining and laser-scanning confocal microscopy of mitotic NRK cells to analyze whether nuclear membrane proteins are restricted to a subcompartment of the ER during mitosis as they are during interphase. In this study, we compared the localization of proteins of both the inner nuclear membrane and nuclear pore membrane to the distribution of bulk ER membranes. The ER was detected with two fluorescent dyes that selectively label the ER, DiOC6
(Lee and Chen, 1988
; Terasaki and Reese, 1992
) and R6 (Terasaki and Reese, 1992
), as well as with several polyclonal antibodies against ER proteins (see below).
and a polyspecific anti-ER antibody raised against salt and EDTA-extracted rough microsomes (Louvard et al., 1982
) gave essentially coincident patterns of fluorescence labeling in interphase and metaphase NRK cells (Fig. , A
, respectively), where arrays of tubular and vesicular membrane elements were seen in the optical sections. DiOC6
and a polyclonal antibody against protein disulfide isomerase, an abundant soluble protein of the ER lumen (Vaux et al., 1990
), also gave nearly coincident labeling patterns in interphase and metaphase cells (Fig. , C
, respectively). Similar results were obtained by double labeling of cells with DiOC6
and an antibody against another major soluble protein of the ER lumen, calnexin (Ware et al., 1995
; data not shown). Finally, both DiOC6
and R6 gave coincident staining patterns in interphase and metaphase cells (Fig. , E
, respectively). These data indicate that under our staining conditions, DiOC6
, R6, and the polyspecific anti-ER antibody selectively label the ER in NRK cells. We have used these three staining reagents interchangeably to detect the ER throughout this study.
Figure 1 Localization of ER membranes with antibodies and fluorescent dyes. NRK cells were fixed and labeled for indirect immunofluorescence microscopy with a polyspecific anti-ER antibody (A and B) or anti–protein disulphide isomerase antibody (anti-PDI (more ...)
We next compared the localizations of LAP1, an integral protein of the inner nuclear membrane, and ER membranes during interphase and mitosis (Fig. ). In interphase cells, LAP1 was localized in a nuclear rim pattern, whereas the ER membranes were seen as an extensive reticular/vesicular network extending throughout the cell (Fig. A). As expected, overlap between interphase NE and ER labeling occurred at the nuclear rim (Fig. A, Merge). By prometaphase, when the NE has disassembled, LAP1 appeared to be localized throughout the entire ER; nearly every membrane structure labeled with the ER probe also was labeled with the LAP1 antibody (Fig. B, compare LAP1 and ER). Moreover, there was a roughly uniform distribution of LAP1 throughout ER membranes (i.e., the relative intensity of labeling of the various membrane structures is similar with both the LAP1 and ER probes). Dispersion of LAP1 throughout ER membranes appears to occur very close to the time of nuclear lamin depolymerization, since all late prophase cells that we examined by double immunofluorescence staining contained LAPs and lamin in the portions of the NE that remained assembled (Yang, L., and L. Gerace, unpublished observations).
Figure 2 Comparison of the localizations of LAP1 and ER membranes throughout mitosis. NRK cultures enriched in mitotic cells were fixed and examined by double immunofluorescence microscopy after labeling with the LAP1-specific monoclonal antibody RL13 and a (more ...)
The dispersion of LAP1 throughout ER membranes persisted through metaphase (Fig. C) and mid-anaphase (Fig. D, left cell). In late anaphase, when nuclear membranes begin to assemble around chromosomes, LAP1 became segregated from bulk ER membranes at the periphery of the chromosome masses (Fig. D, right cell). Furthermore, in some late anaphase cells where LAP1 association with chromosomes was apparent, the LAP1 remaining in the peripheral ER was not uniformly localized throughout, but appeared to be locally concentrated in certain ER elements (Fig. D, right cell). A similar phenomenon was seen for LAP2 (see below; data not shown). LAP1 was exclusively perinuclear by telophase when it was separated from all peripheral ER membranes (Fig. E). It should be noted that LAP1 has a higher concentration in the telophase NE relative to the ER label than in the interphase NE (i.e., the NE appears green in the merged images of Fig. E, while it appears yellow in the merged images of Fig. A). This very likely is due to a substantial increase in the surface area of the NE from telophase to early G1 without concomitant synthesis of new NE proteins, thereby decreasing the relative LAP1 concentration.
We obtained comparable localization results for LAP1 during mitosis using either a monoclonal antibody to LAP1 (shown in Fig. ) or a polyclonal antibody raised against the nucleoplasmic domain of LAP1 (data not shown; see Materials and Methods), lending confidence to our findings. These results indicate that LAP1 becomes essentially randomized throughout the ER by prometaphase when the NE is disassembled and again becomes concentrated in a discrete subdomain of the ER in late anaphase when the NE reassembles.
We next extended this analysis to LAP2, another integral membrane protein of the inner nuclear membrane, and gp210, an integral protein of the nuclear pore membrane. Similar to the results seen with LAP1, LAP2 and gp210 appeared to be dispersed throughout all ER membranes in mitosis. In prometaphase (data not shown) and metaphase (Fig. ) cells, virtually all ER membranes were labeled in a roughly uniform fashion with antibodies to LAP2 (Fig. A
) and gp210 (Fig. B
). The proteins were resegregated to the nuclear periphery at the end of mitosis (Gerace et al., 1982
; Foisner and Gerace, 1993
). Comparable results on the localization of LAP2 in mitotic cells were obtained using either monoclonal antibodies (shown in Fig. ) or polyclonal antibodies raised against a fragment of the nucleoplasmic domain of this protein (data not shown; see Materials and Methods). As observed previously (Chaudhary and Courvalin, 1993
), we found that assembly of LAP1 and LAP2 around chromosomes, which occurred in late anaphase (Foisner and Gerace, 1993
), preceded the assembly of the majority of gp210 (data not shown).
Figure 3 Comparison of the localization of ER membranes to the distributions of LAP2, gp210, and α-mannosidase II in metaphase cells. NRK cultures enriched in mitotic cells were fixed and examined by double labeling with DiOC6 and the LAP2-specific monoclonal (more ...)
As a control, we analyzed the localization in mitotic cells of α-mannosidase II, an integral membrane protein of the Golgi complex (Velasco et al., 1993
). Golgi membranes are known to remain separate from the ER during mitosis (Warren and Wickner, 1996
) and therefore should present a distribution distinct from ER membranes in confocal microscopy. As expected, in mitotic cells the antibody to α-mannosidase II labeled a set of membrane structures that largely did not overlap with ER membranes, even though both membranes were extensively dispersed throughout the cytoplasm (Fig. C
). This indicates that our microscope procedure would be able to clearly distinguish hypothetical NE-specific membranes, if they existed, as a population separate from bulk ER membranes.
To directly visualize the mitotic dynamics of LAPs, we expressed chimeras consisting of LAP1C and LAP2 fused to green fluorescent protein (GFP) in cultured mammalian cells and examined the GFP fluorescence by confocal light microscopy (Yang, L., and L. Gerace, unpublished observations). The GFP chimeras were localized to the NE in interphase cells and were dispersed throughout ER membranes during mitosis, in agreement with the results of immunofluorescence localization. Unfortunately, because the fluorescence intensity of membrane structures labeled with the GFP–LAP fusion proteins was strongly diminished after the proteins were distributed throughout the ER in mitosis, we were not able to carry out a real time analysis of the mitotic dynamics of LAPs in the NE.
In summary, our results indicate that integral membrane proteins of both the inner nuclear membrane (LAP1 and LAP2) and nuclear pore membrane (g210) are dispersed throughout the ER during mitosis in NRK cells, at the resolution of light microscopy. We have obtained similar results in several other cultured mammalian cell lines (CHO, COS, and HeLa cells; data not shown), and we believe that the phenomenon we have described is a general property of these NE proteins during mitosis.
Localization of NE and ER Membranes in Mitotic Cells by Immunogold Electron Microscopy
To confirm and extend the results we obtained with confocal light microscopy, we carried out double immunogold labeling of digitonin-permeabilized mitotic NRK cells to localize LAP1 and ER membranes at the EM level (Fig. ). The populations used for this analysis were selected from nocodazole-arrested cultures and were highly enriched in metaphase-like cells. ER membranes were labeled with a rabbit polyclonal anti-ER antibody and 5-nm gold coupled to a secondary antibody, and LAP1 was detected with a mouse monoclonal antibody and 10-nm gold coupled to a secondary antibody. The antibody concentrations were adjusted so that similar labeling densities were obtained with the 5- and 10-nm gold particles. Both antibodies labeled two categories of intracellular membrane structures: discrete, relatively large (usually 50–500 nm in diameter) vesicles with an obvious lumen (Fig. A, large arrowheads; Fig. C, top row) and densely-staining aggregates that contained thin tubules and clusters of small vesicles (Fig. A, small arrows; Fig. C, bottom two rows). The density of gold labeling was considerably higher for the latter category of structures than for the former. At least in part, this probably reflects the larger amount of membrane surface per unit area in aggregates of thin tubules and small vesicle clusters as compared to large, single vesicles.
Figure 4 Comparison of the localizations of LAP1 and ER membranes in metaphase cells by double immunogold labeling. Populations of nocodazole-arrested metaphase NRK cells were processed for double immunogold labeling by incubation with the LAP1-specific monoclonal (more ...)
Most membrane structures of both classes (i.e., discrete vesicles and aggregates of thin tubules/small vesicles) that were labeled with anti-ER antibodies also were labeled with anti-LAP1 antibodies (Fig. A, arrows and arrowheads, and gallery in Fig. C). The gold labeling with antiER and -LAP1 antibodies was specific, as very little labeling of membranes was obtained in samples incubated with gold-coupled secondary antibodies alone (Fig. B). Furthermore, very little labeling of the peripheral ER was obtained with anti-LAP1/10-nm gold particles in interphase cells (data not shown), where LAP1 is undetectable in the peripheral ER by immunofluorescence microscopy (Fig. ). Finally, the anti-ER and -LAP1 antibodies labeled only a fraction of all membrane structures in the permeabilized cells (e.g., Fig. A).
We confirmed the close colocalization of the ER and LAP1 probes by quantitative analysis. In one analytical method, a field containing circular windows with a diameter of 100 nm was randomly placed on prints of electron micrographs. We found that 82% of the windows that contained at least two 5-nm gold particles (ER probe) also contained at least one 10-nm particle (LAP1 probe) (n = 55). By contrast, only 3.8% of all random windows contained at least one 10-nm gold particle (n = 500). In a second method of analysis, we measured the distance from each 5-nm gold particle (ER probe) to the nearest 10-nm gold particle (LAP1 probe). We found that 68.2% of all 5-nm gold particles had a 10-nm gold particle localized within a radius of 100 nm (n = 197). Considered together, these results indicate that LAP1 is located close to most of the ER label. If LAP1 were restricted to a minor subset of ER membranes, a much smaller fraction of the ER label would be expected to have closely associated LAP1. In conclusion, the findings from immunogold EM are in close agreement with the results from confocal light microscopy and confirm that LAP1 is dispersed throughout ER membranes during mitosis.
Order of Lamin and LAP Assembly at the End of Mitosis
Our localization studies suggest that binding interactions are likely to be important for localizing integral membrane proteins to the reforming NE at the end of mitosis (see Discussion). In principle, nuclear lamins could provide binding sites to promote this process if lamins were to assemble around chromosomes at the same time as integral membrane proteins. Although recent immunofluorescence localization studies with conventional light microscopy showed that LAP1 and LAP2 (Foisner and Gerace, 1993
) and p58/LBR (Chaudhary and Courvalin, 1993
) become concentrated around chromosomes at the end of mitosis before most lamins, these studies could not exclude the possibility that a fraction of lamins associates with the chromosome surfaces at the same time as the inner membrane proteins. Lamins exist in a large stoichiometric excess over integral membrane proteins of the inner nuclear membrane, and the high concentration of disassembled lamins in the cytosol would make it difficult to detect chromosome-associated lamins by conventional light microscopy (see Gerace and Foisner, 1994
To reinvestigate this question, we carried out double immunofluorescence localization of lamin A and LAPs in cultured NRK cells during late mitosis and examined specimens using confocal light microscopy to enhance the ability to visualize chromosome-associated lamins in the presence of disassembled cytosolic lamins. As shown in Fig. , when LAP1 (A
) and LAP2 (C
) started to become concentrated at parts of the chromosome surfaces in late anaphase, some lamin A also was concentrated in the same regions of the chromosomes. By early telophase, we observed that virtually all LAPs were concentrated at the chromosome surfaces, while a significant fraction of lamins remained unassembled (Foisner and Gerace, 1993
; and data not shown), while by mid-late telophase (Fig. , B
), most of the lamin pool had reassembled as well. These data indicate that lamin and LAPs associate with the chromosome surfaces in late anaphase in a temporally and spatially coordinated fashion, even though much lamin remained unassembled in early telophase when the assembly of LAPs was essentially completed (Foisner and Gerace, 1993
). Thus, even though lamin and LAPs begin to associate with chromosome surfaces at the same time in late anaphase, the half-time of assembly of the lamin pool appears to be longer than that of LAPs.
Figure 5 Comparison of the localization of lamins to the distribution of LAPs at the end of mitosis. NRK cultures enriched in mitotic cells were labeled for double immunofluorescence microscopy with an antibody against lamin A and the LAP1-specific monoclonal (more ...)