Georg Krohne

The nuclear lamins: A multigene family of proteins in evolution and differentiation☆

The nuclear lamina consists of a proteinaceous layer or meshwork situated subjacent to the inner nuclear membrane. It is a karyoskeletal structure formed by a polymer containing one to three major polypeptides collectively termed the lamins. In all cells examined of vertebrates and invertebrates, the lamins exhibit very similar Mr ranging from 60 000 to 80 000. In vertebrates, two groups of lamins can be distinguished by their isoelectric value, one being near-neutral and the other acidic (isoelectric pH values of 5.6 and lower). The lamins represent a family of polypeptides with regions highly conserved during evolution. In certain species, e.g., the amphibian, Xenopus laevis, they exhibit cell type-specific expression during embryonic development, terminal differentiation of certain somatic cells, and gametogenesis. The nuclear lamina of diverse cell types can be composed of one, two or three different lamin polypeptides, without obvious differences in its morphology.

Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution

One of the most complex molecular machines of cells is the nuclear pore complex (NPC), which controls all trafficking of molecules in and out of the nucleus. Because of their importance for cellular processes such as gene expression and cytoskeleton organization, the structure of NPCs has been studied extensively during the last few decades, mainly by electron microscopy. We have used super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) to investigate the structure of NPCs in isolated Xenopus laevis oocyte nuclear envelopes, with a lateral resolution of ~15 nm. By generating accumulated super-resolved images of hundreds of NPCs we determined the diameter of the central NPC channel to be 41±7 nm and demonstrate that the integral membrane protein gp210 is distributed in an eightfold radial symmetry. Two-color dSTORM experiments emphasize the highly symmetric NPCs as ideal model structures to control the quality of corrections to chromatic aberration and to test the capability and reliability of super-resolution imaging methods.

Argyrophilic nuclear and nucleolar proteins of Xenopus laevis oocytes identified by gel electrophoresis

Abstract In order to identify argyrophilic proteins of nuclei and nucleoli, in particular those responsible for the ‘nucleolar Ag staining’ widely used in cytology, we have utilized oocytes of Xenopus laevis because of the abundance of ‘pure’ extrachromosomal nucleoli. Examination of oocytes by light and electron microscopy shows that the large extrachromosomal nucleoli are heavily stained with the Ag technique and that the Ag deposits are largely enriched in, if not exclusive to, the internal, fibrillar region. The same pattern of Ag staining in internal regions of nucleoli is observed in isolated nucleoli from which soluble nuclear proteins were removed by extensive washing. Argyrophilic proteins of isolated oocyte nuclei and purified nucleoli have been identified by reaction with AgNO 3 and formaldehyde on gel-electrophoretically separated polypeptides. Among nuclear proteins, the most prominent argyrophilia is associated with nucleoplasmin, a soluble MW 30000 phosphoprotein of the nuclear sap. In addition, four minor Ag-staining nuclear proteins have been observed. By contrast, the only strongly argyrophilic protein observed on gel electrophoresis of proteins of purified nucleoli is a high molecular weight component (apparent MW 195000) which is often resolved in a characteristic ‘pair’ of closely spaced polypeptide bands. The enrichment of this high molecular weight argyrophilic protein in isolated nucleoli and the corresponding absence of argyrophilic proteins of the nuclear sap, including nucleoplasmin, indicates that this protein contributes to the nucleolus-specific Ag staining observed in histological sections. The possible nature of this polypeptide of MW 195000 is discussed.

Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization

Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod‐shaped Gram‐negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus‐ and species‐specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the γ‐subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the γ‐subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co‐evolution of the symbiotic partners and a vertical transmission of the bacteria.

Interaction of Xenopus lamins A and LII with chromatin in vitro mediated by a sequence element in the carboxyterminal domain

Morphological data suggest an interaction of the nuclear lamina with chromatin which markedly changes during the cell cycle. To study the molecular basis of this interaction we developed a novel lamin/chromatin binding assay that quantitated the binding of soluble, radiolabeled lamins to minichromosomes assembled in Xenopus laevis oocyte nuclear extracts. Lamins were derived from couple in vitro transcription and translation of the corresponding cDNAs. Chromatin binding was detected by monitoring the cofractionation with assembled minichromosomes in gel filtration and sucrose gradient centrifugation. Binding of lamins to chromatin increased with chromatin concentration and was accompanied by lamin polymerization. Lamins of the A-(Xenopus LA and human LC) as well as the B-type (Xenopus LI and LII) showed strikingly different chromatin binding capacities. Lamins A and LII bound efficiently of lamins LI and LC was detected. Using site-directed mutagenesis, we were able to define carboxy-terminal sequence elements of LA and LII required for the observed lamin/chromatin interaction that are rich in serine, threonine, and glycine residues. Competition experiments with a synthetic peptide containing the chromatin binding motif of lamin A corroborate the importance of these sequence elements in the lamin/chromatin interaction.

Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B2), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B2 is coexpressed with lamin B1 (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found thatXenopus laevis lamin LII is the amphibian homolog of mammalian lamin B2. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.

LEM‐Domain Proteins: New Insights into Lamin‐Interacting Proteins

LEM-domain proteins present a growing family of nonrelated inner nuclear membrane and intranuclear proteins, including emerin, MAN1, LEM2, several alternatively spliced isoforms of LAP2, and various uncharacterized proteins in higher eukaryotes as well as the Drosophila-specific proteins otefin and Bocksbeutel. LEM-domain proteins are involved in diverse cellular processes including replication and cell cycle control, chromatin organization and nuclear assembly, the regulation of gene expression and signaling pathways, as well as retroviral infection. Genetic analyses in different model organisms reveal new insights into the various functions of LEM-domain proteins, lamins, and their involvement in laminopathic diseases. All these findings as well as previously proposed ideas and models have been summarized to broaden our view of this exciting protein family.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

A major soluble acidic protein located in nuclei of diverse vertebrate species

Abstract Guinea pig antibodies against the most frequent nuclear protein of oocytes of Xenopus laevis were used for the characterization and localization, by immunoprecipitation and immunofluorescence microscopy, of this or immunologically related protein(s) in tissues and in cultured cells of diverse vertebrates. The antigen present in Xenopus oocyte nuclei was soluble, acidic, and contained polypeptide(s) of molecular weight of about 30000. It appeared with a broad range of isoelectric variants (apparent isoelectric point (IEP) values between 4.7 and 6.0) which was shown to reflect at least partly differences of phosphorylation. A similar protein was also abundant in oocytes of other amphibia but distinct differences in electrophoretic mobility and IEPs were noted between different species. Similar or immunologically related proteins were also detected, by immunofluorescence microscopy, in diverse tissues and cultured cells of other vertebrates (amphibia, birds, mammals). Mammalian cell lines that showed strong nuclear staining with antibodies to the protein of Xenopus oocytes included examples of marsupial (PtK2), murine (mouse 3T3) and human (HeLa) origin. Certain cell types characterized by very low levels of transcription and predominance of condensed chromatin, such as amphibian and avian erythrocytes and late stages of spermiogenesis, appeared to be devoid of the protein. During interphase and meiotic prophase the protein was detected only in the nucleus where it showed a dispersed distribution, sometimes in ‘punctate’ patterns, and no specific enrichment in nucleoli or dense chromatin structures. During nuclear divisions of both mitosis and meiosis the protein was spread throughout the cytoplasm and appeared to be greatly reduced, if not totally absent, in metaphase and anaphase chromosomes. In immunolocalization experiments considerable losses of the protein into the incubation solutions were noted, which could give rise to artifacts of localization, but this effect could be reduced by modifications of preparation conditions. Possible functions of this protein, the determinant of which has been largely conserved in vertebrate evolution and which seems to be similar, if not identical to the ‘nucleosome assembly factor’ protein described in egg homogenates of Xenopus , are discussed, especially in relation to the formation of chromatin and ribonucleoproteins.

The major polypeptides of the nuclear pore complex

Abstract Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [3H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.