Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.     

Towards and understanding of nuclear morphogenesis

In the age of “virtual reality,” the imperfect microscopic silhouettes of cells and organelles are gradually being replaced by calligraphic computer drawings. In this context, textbooks and introductory slides often depict the cell nucleus as a smooth-shaped, featureless object. However, in reality, the nuclei of different cells possess distinct sizes and morphological features which develop in a programmed fashion as each cell differentiates. To dissect this complex morphogenetic process, we need to identify the basic elements that determine nuclear architecture and the regulatory factors involved. Recently, clues about the identity of these components have been obtained both by systematic analysis and by serendipity. This review summarizes a few recent findings and ideas that may serve as a first forum for future discussions and, I hope, for further work on this topic. © 1994 Wiley-Liss, Inc.     

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.     

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.     

Soe features of DNA fragments associatedin vivo with the nuclear lamina

Ehrlich Ascites Tumour cells were irradiated with UV-light to crosslink DNA to proteinsin vivo. The DNA fragments associated with the nuclear lamina were purified and characterized. The results of the Cot analysis and the hybridization experiments suggest that the DNA fragments attached to the nuclear lamina although containing the entire complexity of genomic DNA are enriched in some highly repeated sequences.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

Remodelling of the nuclear periphery during muscle cell differentiation in vitro

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.     

Dynamics of lamin A/C in porcine embryos produced by nuclear transfer

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.     

DNA damage response by single-strand breaks in terminally differentiated muscle cells and the control of muscle integrity

DNA single-strand breaks (SSB) formation coordinates the myogenic program, and defects in SSB repair in post-mitotic cells have been associated with human diseases. However, the DNA damage response by SSB in terminally differentiated cells has not been explored yet. Here we show that mouse post-mitotic muscle cells accumulate SSB after alkylation damage, but they are extraordinarily resistant to the killing effects of a variety of SSB-inducers. We demonstrate that, upon SSB induction, phosphorylation of H2AX occurs in myotubes and is largely ataxia telangiectasia mutated (ATM)-dependent. However, the DNA damage signaling cascade downstream of ATM is defective as shown by lack of p53 increase and phosphorylation at serine 18 (human serine 15). The stabilization of p53 by nutlin-3 was ineffective in activating the cell death pathway, indicating that the resistance to SSB inducers is due to defective p53 downstream signaling. The induction of specific types of damage is required to activate the cell death program in myotubes. Besides the topoisomerase inhibitor doxorubicin known for its cardiotoxicity, we show that the mitochondria-specific inhibitor menadione is able to activate p53 and to kill effectively myotubes. Cell killing is p53-dependent as demonstrated by full protection of myotubes lacking p53, but there is a restriction of p53-activated genes. This new information may have important therapeutic implications in the prevention of muscle cell toxicity.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly. Project supported by the National Natural Science Foundation of China.     

Poly(ADP-ribose) polymerase-1: association with nuclear lamins in rodent liver cells

The distribution of poly(ADP-ribose) polymerase-1 (PARP-1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A- and B-type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix-associated PARP-1 partitioned, in an active form, with the insoluble, lamin-enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP-1-protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP-1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X-100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co-immunoprecipitation experiments our results show that the lamins are prominent PARP-1-binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.     

DNA replication and nuclear architecture

The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.     

Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.     

Co-localization of PARP-1 and lamin B in the nuclear architecture: a halo-fluorescence- and confocal-microscopy study

A functional interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and lamin B has recently been proposed by nuclear fractionation, crosslinking, and immunoprecipitation experiments. Here we use fluorescence microscopy to verify and extend these findings. We analyze nuclear halo preparations by fluorescence in situ immuno staining (FISIS), which shares attributes with traditional nuclear fractionation techniques, and by confocal laser scanning microscopy (CLSM). The results agree in that a major part of the enzyme co-localizes with lamin B under physiological conditions, where PARP-1 only has basal activity. After DNA damage and the associated activation of PARP-1, and during the subsequent entry into apoptosis, dramatic changes occur: a gradual release of the enzyme from the lamina, accompanied by its accumulation in nucleoli. Our observations are in line with biochemical evidence for lamin B-PARP-1 interactions under physiological conditions and suggest ways by which these interactions are modified to support PARP-functions in damage and its fate in apoptosis.     

Loss of MT1‐MMP causes cell senescence and nuclear defects which can be reversed by retinoic acid

MT1-MMP (MMP14) is a collagenolytic enzyme located at the cell surface and implicated in extracellular matrix (ECM) remodeling. Mmp14^−/− mice present dwarfism, bone abnormalities, and premature death. We demonstrate herein that the loss of MT1-MMP also causes cardiac defects and severe metabolic changes, and alters the cytoskeleton and the nuclear lamina structure. Moreover, the absence of MT1-MMP induces a senescent phenotype characterized by up-regulation of p16^INK4a and p21^CIP1/WAF1, increased activity of senescence-associated β-galactosidase, generation of a senescence-associated secretory phenotype, and somatotroph axis alterations. Consistent with the role of retinoic acid signaling in nuclear lamina stabilization, treatment of Mmp14^−/− mice with all-trans retinoic acid reversed the nuclear lamina alterations, partially rescued the cell senescence phenotypes, ameliorated the pathological defects in bone, skin, and heart, and extended their life span. These results demonstrate that nuclear architecture and cell senescence can be modulated by a membrane protease, in a process involving the ECM as a key regulator of nuclear stiffness under cell stress conditions.     

Heat-induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo

Membrane-depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low-speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat-shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well-defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat-shock-induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.