The farnesylated nuclear proteins KUGELKERN and LAMIN B promote aging-like phenotypes in Drosophila flies

The nuclear lamina consists of a meshwork of lamins and lamina-associated proteins, which provide mechanical support, control size and shape of the nucleus, and mediate the attachment of chromatin to the nuclear envelope. Abnormal nuclear shapes are observed in aging cells of humans and nematode worms. The expression of laminΔ50 , a constitutively active lamin A splicing variant in Hutchinson–Gilford progeria syndrome patients, leads to the lobulation of the nuclear envelope accompanied by DNA damage, and loss of heterochromatin. So far, it has been unclear whether these age-related changes are laminΔ50 specific or whether proteins that affect nuclear shape such as KUGELKERN or LAMIN B in general play a causative role in senescence. Here we show that in adult Drosophila flies, the size of the nuclei increases with age and the nuclei assume an aberrant shape. Moreover, induced expression of the farnesylated lamina proteins Lamin B and Kugelkern cause aberrant nuclear shapes and reduce the lifespan of adult flies. The shorter lifespan correlates with an early decline in age-dependent locomotor behaviour. Expression of kugelkern or lamin B in mammalian cells induces a nuclear lobulation phenotype in conjunction with DNA damage, and changes in histone modification similar to that found in cells expressing laminΔ50  or in cells from aged individuals. We conclude that lobulation of the nuclear membrane induced by the insertion of farnesylated lamina-proteins can lead to aging-like phenotypes.     

Analysis of nuclear lamin isoprenylation in Xenopus oocytes: isoprenylation of lamin B3 precedes its uptake into the nucleus

Protein prenylation is a posttranslational modification involving the covalent attachment of a prenyl lipid to a cysteine at or near the COOH terminus of a protein. It is required for membrane localization and efficient function of a number of cytoplasmic as well as nuclear proteins including the proto-oncogenic and activated forms of Ras. Farnesylation in conjunction with a nuclear localization signal has been shown to be necessary to target newly synthesized nuclear lamins to the inner nuclear envelope membrane. It is, however, not clear where in the cell isoprenylation of nuclear lamins takes place. In this study we describe in vivo and in vitro experiments on the isoprenylation of the Xenopus oocyte nuclear lamin B3. We show by kinetic analysis that newly synthesized lamins are isoprenylated in the cytosol of oocytes before uptake into the nucleus. From our data it can be concluded that isoprenylation of lamins in the nucleus, as it is observed under certain conditions of isoprene starvation, represents a default pathway rather than the physiological situation. We further analyzed the capacity of isolated nuclei to carry out isoprenylation of B3. Our results are in line with a dual localization of a protein farnesyltransferase in the cytosol and nuclei of amphibian oocytes. Implications for the possible functions of a nuclear protein farnesyltransferase as well as possible mechanisms of the selective inhibition of farnesylation of cytoplasmic proteins by peptidomimetics are discussed.     

Nuclear formation in a Drosophila cell-free system

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.     

Overexpression of the lamina proteins Lamin and Kugelkern induces specific ultrastructural alterations in the morphology of the nuclear envelope of intestinal stem cells and enterocytes

The nuclear envelope has a stereotypic morphology consisting of a flat double layer of the inner and outer nuclear membrane, with interspersed nuclear pores. Underlying and tightly linked to the inner nuclear membrane is the nuclear lamina, a proteinous layer of intermediate filament proteins and associated proteins. Physiological, experimental or pathological alterations in the constitution of the lamina lead to changes in nuclear morphology, such as blebs and lobulations. It has so far remained unclear whether the morphological changes depend on the differentiation state and the specific lamina protein. Here we analysed the ultrastructural morphology of the nuclear envelope in intestinal stem cells and differentiated enterocytes in adult Drosophila flies, in which the proteins Lam, Kugelkern or a farnesylated variant of LamC were overexpressed. Surprisingly, we detected distinct morphological features specific for the respective protein. Lam induced envelopes with multiple layers of membrane and lamina, surrounding the whole nucleus whereas farnesylated LamC induced the formation of a thick fibrillary lamina. In contrast, Kugelkern induced single-layered and double-layered intranuclear membrane structures, which are likely be derived from infoldings of the inner nuclear membrane or of the double layer of the envelope.     

Farnesylated Nuclear Proteins Kugelkern and Lamin Dm0 Affect Nuclear Morphology by Directly Interacting with the Nuclear Membrane

Nuclear shape changes are observed during a variety of developmental processes, pathological conditions, and ageing. The mechanisms underlying nuclear shape changes in the above-mentioned situations have mostly remained unclear. To address the molecular mechanism behind nuclear shape changes, we analyzed how the farnesylated nuclear envelope proteins Kugelkern and lamin Dm0 affect the structure of the nuclear membrane. We found that Kugelkern and lamin Dm0 affect nuclear shape without requiring filament formation or the presence of a classical nuclear lamina. We also could show that the two proteins do not depend on a group of selected inner nuclear membrane proteins for their localization to the nuclear envelope. Surprisingly, we found that farnesylated Kugelkern and lamin Dm0 protein constructs change the morphology of protein-free liposomes. Based on these findings, we propose that farnesylated proteins of the nuclear membrane induce nuclear shape changes by being asymmetrically inserted into the phospholipid bilayer via their farnesylated C-terminal part.     

Nuclear morphology: when round kernels do the Charleston

New studies in Drosophila have identified a novel nuclear envelope protein with a farnesyl moiety, termed Kugelkern/Charleston, that helps regulate the size, shape and position of cellular blastoderm nuclei.     

Developmental control of nuclear morphogenesis and anchoring by charleston, identified in a functional genomic screen of Drosophila cellularisation

Morphogenesis of epithelial tissues relies on the precise developmental control of cell polarity and architecture. In the early Drosophila embryo, the primary epithelium forms during cellularisation, following a tightly controlled genetic programme where specific sets of genes are upregulated. Some of them, for example, control membrane invagination between the nuclei anchored at the apical surface of the syncytium. We used microarrays to describe the global programme of gene expression underlying cellularisation and identified distinct classes of upregulated genes during this process. Fifty-seven genes were then tested functionally by RNAi. We found six genes affecting various aspects of cellular architecture: membrane growth, organelle transport or organisation and junction assembly. We focus here on charleston (char), a new regulator of nuclear morphogenesis and of apical nuclear anchoring. In char-depleted embryos, the nuclei fail to maintain their elongated shape and, instead, become rounded. In addition, together with a disruption of the centrosome-nuclear envelope interaction, the nuclei lose their regular apical anchoring. These nuclear defects perturb the regular columnar organisation of epithelial cells in the embryo. Although microtubules are required for both nuclear morphogenesis and anchoring, char does not control microtubule organisation and association to the nuclear envelope. We show that Char is lipid anchored at the nuclear envelope by a farnesylation group, and localises at the inner nuclear membrane together with Lamin. Our data suggest that Char forms a scaffold that regulates nuclear architecture to constrain nuclei in tight columnar epithelial cells. The upregulation of Char during cellularisation and gastrulation reveals the existence of an as yet unknown developmental control of nuclear morphology and anchoring in embryonic epithelia.     

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

A Comparative Study of Drosophila and Human A-Type Lamins

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm₀, which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction.     

Internuclear exchange of an inner nuclear membrane protein (p55) in heterokaryons: in vivo evidence for the interaction of p55 with the nuclear lamina

The movement between nuclei of an integral protein of the inner nuclear membrane has been studied in rat/mouse and rat/hamster heterokaryons. This protein, p55, was found to equilibrate between nuclei over a period of approximately 6 h in the absence of new protein synthesis. When rat/mouse heterokaryons were constructed using an undifferentiated murine embryonal carcinoma (P19), which lacks lamins A and C, no accumulation of p55 in the mouse cell nucleus was observed. However, P19 nuclei could be rendered competent to accumulate p55 by transfecting the parent cells with human lamin A before cell fusion, supporting the notion that p55 may interact with the nuclear lamina. Since p55 does not appear to be able to dissociate from the nuclear membrane, it is concluded that this exchange between nuclei does not occur in the aqueous phase and instead is probably membrane mediated. It is proposed that this protein may be free to move between the inner and outer nuclear membranes via the continuities at the nuclear pore complexes and that transfer between nuclei occurs via lateral diffusion through the peripheral ER, which appears to form a single continuous membrane system in these heterokaryons. One implication of these observations is that accumulation of at least some integral proteins in the inner nuclear membrane may be mediated by interactions with other nuclear components and may not require a single defined targeting sequence.     

Nonlamin components of the lamina: a paucity of proteins.

Current models of nuclear organization propose that nuclear functions are modulated in part by reversible tethering of chromatin loops to structural elements of the nucleoplasm and the nuclear envelope. Lamins are the best-characterized proteins of the lamina portion of the nuclear envelope and are involved in binding chromatin to the inner nuclear membrane. However, they are not a universal feature of eukaryotic nuclei and do not account fully for the putative functions of the lamina in all organisms. It is possible that nonlamin components of the lamina may substitute for lamins in organisms from which they are absent and modify the properties of lamins during development and the cell cycle. We review the properties of the relatively small number of such components that have been reported, including the young arrest (fs(1)Ya) protein of Drosophila, statin, circumferin, and the MAN antigens. The experimental evidence indicates they are a diverse group of proteins, and that at least some have the potential to modulate the interactions of chromatin, lamins, and the nuclear membranes.     

Dynamic changes in the distribution of cytoplasmic myosin during Drosophila embryogenesis

Dramatic changes in the localization of conventional non-muscle myosin characterize early embryogenesis in Drosophila melanogaster. During cellularization, myosin is concentrated around the furrow canals that form the leading margin of the plasma membrane as it plunges inward to package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific anti-myosin staining at the apical ends of those cells that change shape in regions of invagination. Both of these localizations appear to result from a redistribution of a cortical store of maternal myosin. In the preblastoderm embryo, myosin is localized to the egg cortex, sub-cortical arrays of inclusions, and, diffusely, the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeletal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These distributions are observed with both whole immune serum and affinity-purified antibodies directed against Drosophila non-muscle myosin heavy chain. They are not detected in embryos stained with anti-Drosophila muscle myosin antiserum or with preimmune serum. Although immunolocalization can only suggest possible function, these myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis.     

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.     

Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis

Background  

The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation.     

The CaaX motif is required for isoprenylation, carboxyl methylation, and nuclear membrane association of lamin B2

Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.