Phorbol esters induce transient changes in the accessibility of the carboxy-terminal domain of nuclear lamin A.

Treatment of human epithelial cells in culture with phorbol esters (TPA) gives rise to a transient and reversible loss of accessibility to antibodies of the nonhelical carboxy-terminal domain of nuclear lamin A that distinguishes it from lamin C. No change in the accessibility of epitopes present in the common domain of lamins A and C was observed. Loss of accessibility of lamin A was not due to proteolytic degradation nor to modification of the isoelectric point of lamin A and did not depend upon protein kinase C activation nor protein synthesis. Perturbation of desmosome organization by growth in low calcium blocked the effect of TPA on lamin A. Prolonged exposure to nocodazole, one of the effects of which is a perinuclear collapse of intermediate filaments, also blocked the effect of TPA on lamin A. These results suggest that the initial target of TPA may be at the level of cell-cell contacts and that the perturbation induced by TPA may be propagated via the structural link formed by intermediate filaments between the cell surface and the nucleus, giving rise to a change in conformation of the carboxy-terminal domain of lamin A or to an interaction of this domain with another nuclear component. These results form the basis for the hypothesis that the interphase nuclear lamina may play an active role in the process of mechanochemical signal transduction.     

Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Structure of the globular tail of nuclear lamin

The nuclear lamins form a two-dimensional matrix that provides integrity to the cell nucleus and participates in nuclear activities. Mutations in the region of human LMNA encoding the carboxyl-terminal tail Lamin A/C are associated with forms of muscular dystrophy and familial partial lipodystrophy (FPLD). To help discriminate tissue-specific phenotypes, we have solved at 1.4-A resolution the three-dimensional crystal structure of the lamin A/C globular tail. The domain adopts a novel, all beta immunoglobulin-like fold. FPLD-associated mutations cluster within a small surface, whereas muscular dystrophy-associated mutations are distributed throughout the protein core and on its surface. These findings distinguish myopathy- and lipodystrophy-associated mutations and provide a structural framework for further testing hypotheses concerning lamin function.     

Coated pits in interphase and mitotic A431 cells

Endocytosis is inhibited during mitosis in A431 cells (Warren et al., 1984) but the site of inhibition is unknown. A quantitative method measuring the extent of budding was used to compare coated pits in interphase and mitotic cells. Every stage of budding found in interphase cells was also found in cells at every stage of mitosis. Flatter coated pits appeared more frequent in mitotic cells but this can be partly, if not entirely, explained by their greater size. We conclude that, if budding is inhibited, inhibition must occur at all stages of the budding process.     

Changes in a nuclear matrix antigen during the cell cycle: interphase and mitotic cells

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin-stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two-dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.     

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.     

Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms

Three isoforms of a single nuclear lamin have been identified in Drosophila. Two, lamins Dm1 and Dm2, are present during interphase and are apparently in equilibrium with each other in vivo. The third, lamin Dmmit, is found in cells that have undergone nuclear envelope breakdown, either during meiosis or mitosis. All three isoforms were purified under nondenaturing conditions using a novel technique of immunoaffinity chromatography and their in vitro activities were examined. Interphase lamins Dm1 and Dm2 can assemble into filaments at physiologic ionic strength; assembly is reversible upon addition of concentrated NaCl. Negative staining of filaments formed in vitro shows long, unbranched bundles approximately 20 nm in diameter. Addition of specific antilamin antibodies blocks in vitro assembly completely. In contrast with lamins Dm1 and Dm2, lamin Dmmit remains soluble at physiologic ionic strength. These observations are consistent with the notion that lamina disassembly in vivo is due, at least in part, to changes in properties of the lamins themselves.     

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells

In animals and yeast, the small GTP-binding protein Ran has multiple functions - it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immunofluorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.     

Amino acid pools in mitotic and interphase HeLa cells

Mitotic HeLa cells collected by shake-off synchronizing procedures were found to have elevated amino acid influx rates into the acid-extractable pool. Combined use of colchicine or chilling before testing abolished the increased uptake; high external concentrations of Mg²⁺ further enhanced it. Telophase and subsequent interphase populations showed a lower uptake rate which remained constant throughout most of the next cycle.     

In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail

Xenopus lamin A and a lamin A mutant lacking the complete 280 amino acid long carboxy-terminal tail were expressed in bacteria and purified from inclusion bodies. Electron microscopic analysis of lamin A dimers revealed that the carboxy-terminal 280 amino acids correspond to the globular domain seen in rotary-shadowed wild-type lamin and that the rodlike domain consists of the short non-helical amino terminus and the alpha-helical region. During reconstitution lamin A dimers first formed polar head to tail aggregates which then associated laterally resulting in paracrystals with periodic repeats of 25 nm. In the mutant, the longitudinal and lateral association of dimers had not been influenced, however, periodic repeats were absent in the filament bundles formed. Thus our data clearly demonstrate that carboxy-terminal tails are localized in light-stained regions of negatively stained paracrystals and that they are responsible for the alternating light dark staining of paracrystals. Fibrils, 2 to 3 nm thick, were a common structural element of paracrystals and filament bundles.     

Differential expression of nuclear lamin proteins during chicken development

By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.     

Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras

Understanding how membrane proteins are targeted to and retained within the nuclear envelope (NE) and the fate of these proteins during NE disassembly/reassembly in mitosis is central for insight into the function of the NE in nuclear organization and dynamics. To address these issues we have attached green fluorescent protein (GFP) to a well-characterized protein of the inner nuclear membrane, lamin B receptor, believed to be one of the major chromatin docking protein in the NE. We have used this construct in a variety of applications, including dual-color GFP time-lapse imaging, to investigate the mechanisms underlying protein targeting to the NE and NE breakdown and reassembly during mitosis. In this review, we present a summary of the results from such studies and discuss the photobleaching and imaging methodology on which they were derived.     

The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.     

Cyclin-dependent kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain

Nuclear lamins maintain the nuclear envelope structure by forming long linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The A11 binding mode is characterized by the antiparallel interactions between coil 1b domains, whereas the A22 binding mode is facilitated by interactions between the coil 2 domains of lamin. The junction between A11- and A22-interacting dimers in the lamin tetramer produces another parallel head–tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers depolymerization of lamin filaments, but the associated mechanisms remain unknown at the molecular level. In this study, we revealed using the purified proteins that phosphorylation by the CDK1 complex promotes disassembly of lamin filaments by directly abolishing the ACN interaction between coil 1a and the C-terminal portion of coil 2. We further observed that this interaction was disrupted as a result of alteration of the ionic interactions between coil 1a and coil 2. Combined with molecular modeling, we propose a mechanism for CDK1-dependent disassembly of the lamin filaments. Our results will help to elucidate the cell cycle–dependent regulation of nuclear morphology at the molecular level.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells

Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.     

Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells

Among a library of monoclonal antibodies (mAbs) recognizing developmental markers in the chick embryo, mAb CC-3 was selected because of its differential immunostaining of mitotic cells. The intracellular distribution of the CC-3 antigen (CC-3a) throughout the cell cycle was visualized by immunolocalization. In interphase cells CC-3a resided in the nucleus and was arranged in distinct extranucleolar clusters. At prophase, the nuclear reactivity of CC-3a considerably increased and subsequently extended to the cytoplasm at metaphase. From metaphase through anaphase, most of the reactivity was associated with the mitotic apparatus. During cytokinesis CC-3a was detected in the mid-body and also in discrete speckles dispersed throughout the cytoplasm. The initial interphase pattern was then restored in the two daughter nuclei. Immunoblot analysis demonstrated that a 255-kDa phosphoprotein was present only in the interphase nucleus and that a complete new set of phosphoproteins accounted for the mitotic cell reactivity. The binding of CC-3 was dependent on the phosphorylation of its antigens. CC-3a is an evolutionary conserved molecule; it is present in such phylogenetically distant species as Drosophila and humans. Furthermore, the unique behavior of CC-3 on sections of normal, embryonic, and regenerative tissue and in cell culture immunostaining make it a reliable tool to identify mitotic foci.     

Differential effect of pH on solubilization of nuclear lamins A/C and lamin B

Extraction of isolated rat liver nuclear envelopes with 4M urea at various pH values led to differential solubilization of lamin polypeptides. All three lamins A, B and C were solubilized to 70-80% when extraction was performed at pH 8 while only 50-60% of lamins A and C were solubilized at pH 6, leaving lamin B completely insoluble. These results indicated that the interaction of lamin B with the nuclear envelope was strongly dependent upon the ionization state of lamin B and/or of its putative attachment site.     

A monoclonal antibody which recognises each of the nuclear lamin polypeptides in mammalian cells

A monoclonal IgM has been characterised which recognises the nuclear lamins in all mammalian cells tested. In immunoblotting experiments using both one- and two-dimensional gels it recognises lamins A, B and C. The common antigenic determinant lies on a proteolytic fragment of 46,000 daltons which can be generated from each lamin polypeptide by treatment with chymotrypsin. In immunofluorescence experiments on whole cells and thin frozen sections, the antibody labelled only the nuclear envelope and not the nuclear interior. During mitosis, labelling was found dispersed throughout the cell cytoplasm. By immunoelectron microscopy using the antibody and protein A-gold, only the nucleoplasmic side of the nuclear envelope (the nuclear lamina) was labelled, but there was no labelling of the nuclear pores.