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.     

Characterization of human autoantibodies specific for lamin A

We have characterized human autoimmune polyclonal antibodies reactive with lamin A, a 74 kDa peripheral protein of the nuclear envelope. Unlike other known antibodies to lamin A, the antibodies described here do not crossreact with the structurally related lamin C. These antibodies feature only chi light chains suggesting that their specificity is restricted to a limited number of epitopes. Based on the known amino acid sequence of human lamins A and C, the epitope(s) are most likely located in the 80 amino acid carboxyl tail of mature lamin A.     

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends. Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth. We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p. Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location. On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends. We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.     

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.     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

The apparent absence of lamin B1 and emerin in many tissue nuclei is due to epitope masking

Summary Immunolocalization studies have concluded that the nuclear membrane protein, emerin, is absent from many cell types and that lamin B1 is absent from adult heart and skeletal muscle. We now show that epitope masking in the nucleus is often responsible for failure to detect emerin and lamins in human, rat and pig tissues. Human heart cardiomyocyte nuclei were negative for lamin B1 using a commercial mAb, but were positive using two other lamin B1 antibodies, mAb8D1 and pAbB1-cbs. Rat hippocampal neuronal nuclei were immunostained by mAb8D1, but not pAbB1-cbs, while the commercial antibody stained only a subset. These data suggest that different regions of the lamin B1 molecule are masked in different tissues. Similarly, pig spleen had fewer emerin-positive nuclei than lung (5% vs. 32%), although their emerin content was similar by Western blotting. As mAbs against six epitopes gave the same result, the whole emerin molecule is either masked or redistributed in a subset of cells. Our findings argue that immunostaining evidence can be misleading for expression of nuclear envelope proteins. Problems with lamin B1 immunostaining can be avoided by using mAb8D1, but use of antibodies recognizing different epitopes may reveal cell-specific protein interactions in the nucleus.     

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.     

Association of lamin A/C with muscle gene-specific promoters in myoblasts

The A-type and B-type lamins form a filamentous meshwork underneath the inner nuclear membrane called the nuclear lamina, which is an important component of nuclear architecture in metazoan cells. The lamina interacts with large, mostly repressive chromatin domains at the nuclear periphery. In addition, genome–lamina interactions also involve dynamic association of lamin A/C with gene promoters in adipocytes. Mutations in the human lamin A gene cause a spectrum of hereditary diseases called the laminopathies which affect muscle, cardiac and adipose tissues. Since most mutations in lamin A/C affect skeletal muscle, we investigated lamin–chromatin interactions at promoters of muscle specific genes in both muscle and non-muscle cell lines by ChIP-qPCR. We observed that lamin A/C was specifically associated with promoter regions of muscle genes in myoblasts but not in fibroblasts. Lamin A/C dissociated from the promoter regions of the differentiation specific MyoD, myogenin and muscle creatine kinase genes when myoblasts were induced to differentiate. In the promoter regions of the myogenin and MyoD genes, the binding of lamin A/C in myoblasts inversely correlated with the active histone mark, H3K4me3. Lamin A/C binding on muscle genes was reduced and differentiation potential was enhanced on treatment of myoblasts with a histone deacetylase inhibitor. These findings suggest a role for lamina–chromatin interactions in muscle differentiation and have important implications for the pathological mechanisms of striated muscle associated laminopathies.     

Ectopic overexpression of Drosophila lamin C is stage-specific lethal

To gain insight into the function of the developmentally regulated A-type lamins we transformed Drosophila melanogaster with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C was expressed ectopically after heat shock of embryos and localized to the nucleus. No phenotypic change was observed after lamin C expression in embryos that normally do not contain lamin C. However, ectopic expression of lamin C during most larval (but not pupal) stages stalled growth, inhibited ecdysteroid signaling (in particular during the larval-prepupal transition), resulted in development of melanotic tumors, and finally caused death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity.     

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.     

Repo-Man/protein phosphatase 1 SUMOylation mediates binding to lamin A and serine 22 dephosphorylation

Lamin A phosphorylation/de-phosphorylation is an important process during cells division as it allows for nuclear envelope (NE) disassembly at mitotic entry and its re-assembly during mitotic exit. Several kinases have been identified as responsible for these phosphorylations, but no protein phosphatase has been implicated in their reversal. One of the mitotic phosphosites in lamin A responsible for its dynamic behaviour is serine 22 (S22) which is de-phosphorylated during mitotic exit. Recent evidence has also linked the nuclear pool of lamin A S22ph in interphase to gene expression regulation. Previous work suggested that the phosphatase responsible for lamin A S22 de-phosphorylation is chromatin bound and interacts with lamin A via SUMO-SIM motives. We have previously reported that Repo-Man/protein phosphatase 1 (PP1) is a chromatin-associated phosphatase that regulates NE reformation. Here we propose that Repo-Man/PP1 phosphatase mediates lamin A S22 de-phosphorylation. We indeed show that depletion of Repo-Man leads to NE defects, causes hyperphosphorylation of lamin A S22 that can be rescued by a wild-type but not a SUMOylation-deficient mutant. Lamin A and Repo-Man interact in vivo and in vitro, and the interaction is mediated by SUMOylation. Moreover, the localization of Repo-Man/PP1 to the chromatin is essential for lamin A S22 de-phosphorylation.     

Matrix remodeling controls a nuclear lamin A/C-emerin network that directs Wnt-regulated stem cell fate

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Immunolocalization of ecto-5'-nucleotidase in the kidney by a monoclonal antibody

A monoclonal antibody IgG, has been raised against ecto-5'-nucleotidase purified from rat kidney homogenate. The specificity of the antibody was verified by immunoprecipitation. The distribution of the corresponding antigen in the rat kidney was studied by immunocytochemistry (FITC and PAP technique) in 1 micron thick cryostat sections. The antibody reacted with the brush border of proximal tubules, the apical cell membrane and the apical cytoplasm of intercalated cells in connecting tubules and collecting ducts and with interstitial cells of the cortex. Among the interstitial cells exclusively stellate shaped fibroblasts were reactive whereas rounded interstitial cells (type II interstitial cells) as well as pericytes and endothelial cells of peritubular capillaries were unreactive. Compared to the staining intensity of the fibroblasts in the cortical labyrinth the reactivity of the fibroblasts in the medullary rays of the cortex was weak or absent. Interstitial cells of the entire medulla were unreactive. Concerning the fibroblasts in the periarterial connective tissue, those surrounding the larger arteries (arcuate arteries, cortical radial arteries) were negative, those alongside afferent and efferent arterioles were positive. Endothelia of lymphatic capillaries travelling within the periarterial connective tissue were also positive. All components of the juxtaglomerular apparatus were negative. The findings are consistent with an interstitial production of adenosine, available extracellularly and thus being able to reach the major target sites of adenosine, the smooth muscles of glomerular arterioles, including the granular cells at the glomerular vascular pole.