Spann TP et al. (1997), Disruption of nuclear lamin organization alters...

XB-ART-16746

J Cell Biol 1997 Mar 24;1366:1201-12. doi: 10.1083/jcb.136.6.1201.

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Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

Spann TP , Moir RD , Goldman AE , Stick R , Goldman RD .

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The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

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Species referenced: Xenopus laevis
Genes referenced: lmnb3 mcm3 pcna slc19a1

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	Figure 2. Double label immunofluorescence showing nuclear lamin patterns in a normal interphase BHK cell and in a cell injected with ÎNLA. Nuclei were stained for lamins A/C (LA) (a and c) or lamin B (LB) (b and d). In the uninjected cell, there is a distinctive lamin rim as well as less intense nucleoplasmic foci (a and b), as previously described (39). In cells fixed 2 h after injection, the lamin rim is no longer obvious, and the lamin staining for both lamins A/C and B appears mainly in the same foci (c and d). Confocal optics showing sections through the mid-region of the nucleus. Bar, 5 Î¼m.
	Figure 3. Characterization of nuclei assembled in reactions containing human LA. (a and b) Nucleus stained for (a) human lamin A and (b) Xenopus LB3. The human LA does not disrupt the lamin network and appears to colocalize with the endogenous LB3. (c) Confocal microscopic image of a nucleus after nuclear matrix extraction and staining for human LA. LA staining is retained in the peripheral lamina after the matrix extraction protocol. (dâf) Fluorescence images from a triple label preparation of the same nuclei. (d) Nucleus stained for human lamin A; (e) biotinylated dUTP incorporation pattern as shown by Texas redâconjugated streptavidin (REP); (f) Hoechst dye showing the location of DNA. The incorporation of lamin A does not appear to alter DNA replication in the extract. Bar, 5 Î¼m.
	Figure 6. The addition of ÎNLA to nuclear assembly reactions inhibits DNA replication as shown by the reduced incorporation of biotinylated dUTP. Fluorescence images of the LB3 pattern and biotinylated dUTP incorporation in (a and b) control nuclei and (câf) nuclei formed in the presence of ÎNLA. (a, c, and e) Immunofluorescence using the antibody against Xenopus LB3; (b, d, and f) Texas redâconjugated streptavidin shows the patterns of incorporation of biotinylated dUTP. Disruption of lamin organization greatly inhibited the incorporation of biotinylated nucleotide when compared with control nuclei. However, all of the lamin-disrupted nuclei do contain a faint punctate nucleoplasmic pattern of labeled nucleotide incorporation that is readily resolved by confocal microscopy. (aâd) Conventional optics (see f). (e and f) Confocal optics. Bar, 5 Î¼m.
	Figure 4. Double label fluorescence observations of nuclei stained for different aspects of nuclear envelope structure and function. (aâf) Nuclei were assembled in interphase extracts containing: (a and b) buffer control, (c and d) lamin A, and (e and f) ÎNLA. Nuclei were stained for (a) lamin B3 or (c and e) human lamin A, and (b, d, and f) the nuclear pore WGA binding proteins using fluorescently tagged WGA. Nuclei assembled under all three conditions appear to have essentially normal distributions of WGA binding proteins at the nuclear periphery. (g and h) Nucleus assembled in the presence of ÎNLA and stained for (g) ÎNLA and (h) the membrane dye DIOC6 (MEM). The nucleus contains a disrupted lamin organization but retains normal membrane staining. Bar, 5 Î¼m. (i and j) Import of wild-type lamin A into (i) buffer control and (j) ÎNLA-disrupted nuclei. The wildtype lamin A was detected using the myc 9E10 epitope antibody (13). Nuclei were assembled with or without ÎNLA, and 90 min after the initiation of assembly, myc-tagged human lamin A was added to the reaction. The nuclei were fixed 20 min later and stained with the myc antibody. Both (i) control and (j) ÎNLAdisrupted nuclei show prominent myc staining, demonstrating that the disrupted nuclei retain the ability to import protein. The majority of the imported protein localizes to the characteristic foci of ÎNLA-disrupted nuclei. Confocal optics showing sections through the mid-region of nuclei. Bar, 5 Î¼m.
	Figure 5. Nuclei assembled in an interphase extract containing ÎNLA. (aâc) Conventional fluorescence images of lamin and DNA patterns of a nucleus stained for (a) human LA, (b) Xenopus LB3, and (c) DNA. (d and e) Confocal images of a disrupted nucleus stained for (d) human LA and (e) Xenopus LB3. The endogenous lamin structure has been disrupted and LB3 appears in foci colocalizing with ÎNLA. Bar, 5 Î¼m.
	Figure 7. The addition of ÎNLA to nuclear assembly reactions reduces [32P]dCTP incorporation by â¼95%. (A) Autoradiogram of an agarose gel showing the incorporation of 32P-labeled dCTP into the DNA of nuclei formed in the presence of buffer control or ÎNLA. After nuclear assembly, the samples were treated as described in Materials and Methods and resolved on an 0.8% agarose gel. The upper band is at the origin of the gel. (B) Quantitation of replication assays shown in A. The radioactive signal of the dried gel was quantitated with a FUJIX BAS 2000 phosphoimager. The sum of the signal intensity/area value for both bands in each lane was used to measure the total incorporation of radioactivity into DNA. The average value for four replicate assays was plotted in a bar graph, where the vertical axis represents the signal/area values (in thousands) determined by the imager. The average value for samples containing ÎNLA was 2,292, with values ranging from 2,035â2,513. The average value for the control samples was 39,030, with samples ranging from 34,514â47,443. The addition of ÎNLA to the nuclear assembly reaction reduced the incorporation of 32P-labeled dCTP to â¼5% of that found in control reactions.
	Figure 8. Nuclei formed in the presence of ÎNLA, and then subsequently stained for ÎNLA or lamin B3 and one of several early DNA replication markers. (a and b) Nucleus stained for (a) ÎNLA and (b) DNA polymerase Î±. (c and d) Nucleus stained for (c) LB3 and (d) XORC2. (e and f) Nucleus stained for (e) LB3 and (f) XMCM3. The distribution of DNA polymerase Î±, XORC2, and XMCM3 is not altered by the disruption of the lamin structure. Confocal microscopic images showing sections through the middle of the nuclei. Bar, 5 Î¼m.
	Figure 9. Staining patterns of lamin and DNA replication factors involved in elongation in nuclei assembled in the presence of (a and d) buffer or (b, c, e, and f) ÎNLA. (a) Control nucleus stained for PCNA. (b and c) Nucleus assembled in the presence of ÎNLA stained for (b) PCNA and (c) ÎNLA. (d) Control nucleus stained for RFC. (e and f) Nucleus assembled in the presence of ÎNLA stained for (e) RFC and (f) ÎNLA. PCNA and RFC distributions are altered from the control as a consequence of lamin disruption such that PCNA and RFC colocalize with lamin aggregates in these nuclei. Confocal microscope showing sections through the middle of the nuclei. Bar, 5 Î¼m.
	Figure 10. (a and b) Lamin and biotinylated dUTP incorporation in a nucleus formed in the presence of ÎNLA, and subsequently transferred to an interphase extract containing biotinylated dUTP but lacking ÎNLA (see text). Confocal micrographs showing sections through the middle of the nucleus. (a) Nucleus stained for Xenopus LB3. (b) Pattern of biotinylated dUTP incorporation as shown by binding of Texas redâconjugated streptavidin. The disrupted nuclei were transferred to a nuclear assembly reaction lacking ÎNLA, where they form a lamin rim and replicate DNA. However, some lamin foci remain. (c) Postassembly disruption of the lamin structure of an in vitro assembled nucleus. ÎNLA was added 90 min after the onset of nuclear formation, a point at which the nuclei have normal lamin organization and have largely completed DNA replication. The addition of ÎNLA disrupts the assembled LB3 staining pattern. Bar, 5 Î¼m.

References [+] :

Adachi, Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. 1992, Pubmed, Xenbase